Effector-deficient anti-CD32A antibodies

ABSTRACT

Effector-deficient anti-CD32a monoclonal antibodies are encompassed, as are method and uses for treating CD32a-mediated diseases and disorders, including, thrombocytopenia, allergy, hemostatic disorders, immune, inflammatory, and autoimmune disorders.

SEQUENCE LISTING

The instant application contains a Sequence Listing, which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 20, 2014, isnamed 01119-0008-00US_SL.txt and is 111,245 bytes in size.

FIELD

Methods and compositions for treating and preventing diseases anddisorders mediated by CD32a are provided.

BACKGROUND

The effector, or Fc, regions of antibodies bind to various receptors onmany different cell types. One such receptor is the CD32a IgG receptor(also known as FcgammaRIIa). It has been reported that human plateletsand other human cells, such as basophils, eosinophils, monocytes,neutrophils, dendritic cells, macrophages, and mast cells, display cellsurface CD32a receptors (Hogarth P M et al. Fc receptor-targetedtherapies for the treatment of inflammation, cancer and beyond (March2012) Nat Rev Drug Discov 11:311; PubMed ID: 22460124; Bruhns P.Properties of mouse and human IgG receptors and their contribution todisease models (June 2012) Blood 119:5640; PubMed ID: 22535666).Activation of CD32a by Fc regions of IgG antibodies (regardless ofantigen specificity) results in a number of in vivo reactions, many ofwhich have negative consequences for the human host. For example, IgGactivation of CD32a can contribute to fatality in heparin-inducedthrombocytopenia (HIT; see Boon D M et al. Heparin-inducedthrombocytopenia and thrombosis: a potential fatal complication in aroutine treatment (March 1995) Neth J Med 46:146; PubMed ID: 7731489;and Warkentin T E et al. Sera from patients with heparin-inducedthrombocytopenia generate platelet-derived microparticles withprocoagulant activity: an explanation for the thrombotic complicationsof heparin-induced thrombocytopenia (December 1994) Blood 84:3691;PubMed ID: 7949124). It has also been reported that IgG-mediatedactivation of CD32a on neutrophils, monocytes, and macrophages promotesairway inflammation, allergic reactions, and anaphylaxis. See, e.g.Jönsson F. et al. Human Fc-gamma-RIIA induces anaphylactic and allergicreactions (2012 Mar. 15) Blood 119:2533-44, PubMed ID: 22138510.Activation of CD32a by IgG-Fc can also contribute to thrombosis in HIT(see, e.g. Arepally G et al. Fc gamma RIIA HSR 131 polymorphism,subclass-specific IgG anti-heparin/platelet factor 4 antibodies andclinical course in patients with heparin-induced thrombocytopenia andthrombosis (January 1997) Blood 89:370; PubMed ID: 9002937; Newman P Met al. Heparin-induced thrombocytopenia: new evidence for the dynamicbinding of purified anti-PF4-heparin antibodies to platelets and theresultant platelet activation (July 2000) Blood 96:182; PubMed ID:10891449; Jaffray B et al. Fatal venous thrombosis after heparin therapy(March 1991) Lancet 337:561; PubMed ID: 1671929).

In a 2012 report by Jönsson et al., the authors reported that blockingthe CD32a receptor protected mice from local and systemic anaphylaxis,and concluded that “[t]argeting Fc[gamma]RIIA with specific blockingmolecules in inflammation and autoimmune/allergic reactions in humansmight lead to similar inhibition as we reported recently for mouseFc[gamma]RIIIA in a murine model of rheumatoid arthritis.” Id. at 2542.Jönsson continued that “[b]locking Fc[gamma]RIIA using divalent ligands(eg, mAb IV.3) to prevent allergic and autoimmune disease in humans,however, should not be envisioned, as we report here that high-doses ofmAb IV.3 induced rather than prevented anaphylaxis.” Id. at 2542(emphasis added). Thus, while blockade of CD32a was a desired goal fortreating inflammatory, autoimmune and allergic disorders, those of skillin the art did not envision blockade with CD32a antibodies due to theirknown negative side effects upon in vivo administration. The inventorshave now solved this problem by providing novel CD32a antibodies that donot elicit negative side effects such as anaphylaxis.

In addition to diseases and disorders mediated by activation of CD32a, anumber of diseases and disorders are mediated by CD32a interactions withthe Fc regions of immobilized IgG, which do not directly activate CD32a.“Immobilized IgG” refers to antibody molecules that are bound to, orprecipitated on, a surface and thus have restricted mobility (i.e., are“immobilized”). Cells having immobilized IgG may alternatively bedescribed as “IgG-coated” cells. CD32a is known to interact only weaklywith the Fc region of single IgG molecules, whether soluble (Hogarth P Met al. Fc receptor-targeted therapies for the treatment of inflammation,cancer and beyond (March 2012) Nat Rev Drug Discov 11:311; PubMed ID:22460124) or immobilized (Wines B D et al. The IgG Fc contains distinctFc receptor (FcR) binding sites: the leukocyte receptors Fc gamma RI andFc gamma RIIa bind to a region in the Fc distinct from that recognizedby neonatal FcR and protein A (May 2000) J Immunol 164:5313; PubMed ID:10799893). Thus, antibodies incapable of directly activating CD32anevertheless caused CD32a-mediated diseases and disorders such asthrombocytopenia when such antibodies were immobilized on the plateletsurface (McKenie et al. The role of the human Fc receptor FcgammaRIIA inthe immune clearance of platelets: a transgenic mouse model (April 1999)J Immunol 162:4311; PubMed ID: 10201963).

IgG-coated platelets (or other cells) are actively cleared from thecirculating blood. For example, it is well known that in immunethrombocytopenic purpura (ITP), human patients with circulatinganti-platelet antibodies (typically IgG) experience platelet clearancemediated in large part by the spleen and the liver, where Fc-receptors(including CD32a) on phagocytes bind and retain the IgG-coatedplatelets. Removal of the spleen (splenectomy) can alleviate thiscondition. Unlike with HIT, however, thrombosis is not typicallyassociated with the clearance of IgG-coated platelets in ITP; rather,the clinical problem of bleeding is the more prominent concern, andimproved therapeutic strategies for this problem are needed (Altomare Iet al. Bleeding and mortality outcomes in ITP clinical trials: a reviewof thrombopoietin mimetics data (October 2012) Am J Hematol 87:984;PubMed ID: 22729832).

CD32a is also known to mediate clearance of IgG-coated red blood cells(erythrocytes) in CD32a mediated diseases and disorders such asautoimmune hemolytic anemia (AIHA). Targeting CD32a with blocking mAbswould thus seem to be of great utility in treating AIHA; indeed, thiswas reported with the anti-CD32 mAb, MDE-8, which was shown toameliorate IgG antibody-induced anemia in mice having a human CD32atransgene but otherwise lacking classical mouse IgG receptorfunction—that is, the animals used to test MDE-8 lacked functional mouseIgG receptors of type I (CD64) and type III (CD16), leaving open thequestion as to how these might affect MDE-8 activity in vivo (vanRoyen-Kerkhof A et al. A novel human CD32 mAb blocks experimental immunehaemolytic anaemia in FcgammaRIIA transgenic mice (July 2005) Br JHaematol 130:130; PubMed ID: 15982355). MDE-8 has not been developed asa therapeutic antibody. Reasons for the lack of preclinical developmentof MDE-8 have not been publicly disclosed. However, the inventors havenow identified and solved a previously undescribed problem with MDE-8and other anti-CD32a antibodies, namely by modifying them to reducebinding to IgG Fc-receptors, and so that they no longer mediateclearance via CD32a when immobilized on cells, thereby making clinicaldevelopment possible.

Compositions that can prevent CD32a-mediated clearance of IgG-coatedcells without causing negative side effects are therefore desired. Theinventors herein describe such compositions and detail their successfuluse to treat and prevent CD32a-mediated diseases and disorders.

SUMMARY

In accordance with the description, the inventors have discovered thatadministration of native anti-CD32a antibodies in vivo causes adversereactions that include thrombocytopenia, drop in body temperature, andsymptoms of shock. The inventors have found that administeringeffector-deficient anti-CD32a antibodies alleviates these adversereactions.

Therefore, in one embodiment, the present invention provides a methodfor preventing adverse reactions caused by administration of anti-CD32aantibodies by administering effector-deficient anti-CD32a antibodies.Similarly, methods for treating CD32a-mediated diseases or disorderscomprising administering effector-deficient CD32a antibodies areprovided.

In some instances, the CD32a-mediated disease or disorder isthrombocytopenia.

In other embodiments, the CD32a-mediated disease or disorder is asymptom of shock, including anaphylactic shock.

In still further embodiments, the CD32a-mediated disease or disorder isan inflammatory, immune, or autoimmune disease or disorder, includingrheumatoid arthritis (RA), psoriasis, psoriatic arthritis, inflammatorybowel disease, osteoarthritis, and systemic lupus erythematous (SLE).

In still further embodiments, the CD32a-mediated disease or disorder isheparin-induced thrombocytopenia (HIT), immune thrombocytopenic purpura(ITP), antiphospholipid syndrome (APS), thrombosis or thrombocytopeniaassociated with autoimmunity or with certain drugs (e.g., heparin) andantibody therapies (e.g., anti-VEGF, anti-TNFalpha, anti-IgE, oranti-CD40L immunotherapies), transfusion or organ transplantationreactions, viral infection, bacterial infection, allergic asthma,allergic rhinitis, lupus nephritis, antibody-mediated anemia,anaphylaxis, chronic idiopathic urticaria (CIU), or airway inflammation.

The inventors have further discovered that administeringeffector-competent non-anti-CD32a IgG antibodies can cause adversereactions, including thrombocytopenia, a drop in body temperature, orsymptoms of shock. Administering effector-deficient anti-CD32aantibodies prevents these adverse reactions. Therefore, in oneembodiment, the present invention provides a method of administeringeffector-deficient anti-CD32a antibodies to treat adverse reactionscaused by IgG antibodies, including non-CD32a antibodies.

Compounds for use in these methods are provided, includingeffector-deficient chimeric and humanized AT-10 and IV.3 andeffector-deficient human MDE-8 monoclonal IgG antibodies. Theeffector-deficient antibodies comprise at least a portion of the Fcregion, and may be full length or may be truncated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows core body temperatures as a representative measure ofinfusion reaction in CD32A mice injected with either native human MDE-8antibodies (IgG1, IgG2), or two different effector-deficient versions ofthe same MDE-8 antibodies. These effector-deficient antibodies do notcause infusion reactions.

FIG. 2 shows severe thrombocytopenia following intravenous injection ofnative human MDE-8 mAbs into CD32A mice and no thrombocytopeniafollowing intravenous injection of two representative effector-deficienthuman MDE-8 antibodies.

FIG. 3A shows flow cytometric analysis of whole blood from CD32A miceprior to injection of native MDE-8 IgG1 mAbs.

FIG. 3B shows flow cytometric analysis of whole blood from CD32A miceafter intravenous injection of native MDE-8 IgG1 mAbs.

FIG. 3C shows flow cytometric analysis of whole blood from CD32A miceprior to injection of effector-deficient MDE-8 IgG1 mAbs.

FIG. 3D shows flow cytometric analysis of whole blood from CD32A miceafter intravenous injection of effector-deficient MDE-8 IgG1 mAbs.

FIG. 3E shows flow cytometric analysis of whole blood from CD32A miceprior to injection of native MDE-8 IgG2 mAbs.

FIG. 3F shows flow cytometric analysis of whole blood from CD32A miceafter intravenous injection of native MDE-8 IgG2 mAbs.

FIG. 3G shows flow cytometric analysis of whole blood from CD32A miceprior to injection of effector-deficient MDE-8 IgG2 mAbs.

FIG. 3H shows flow cytometric analysis of whole blood from CD32A miceafter intravenous injection of effector-deficient MDE-8 IgG2 mAbs.

FIG. 4A shows flow cytometric analysis of whole blood from CD32A miceafter intravenous injection of IgG immune complexes.

FIG. 4B shows flow cytometric analysis of whole blood fromeffector-deficient MDE-8 IgG1 E269R anti-CD32a mAb pre-treated CD32Amice after intravenous injection of IgG immune complexes.

FIG. 4C shows flow cytometric analysis of whole blood from CD32A miceafter intravenous injection of IgG immune complexes.

FIG. 4D shows flow cytometric analysis of whole blood fromeffector-deficient MDE-8 IgG2 N297A anti-CD32a mAb pre-treated CD32Amice after intravenous injection of IgG immune complexes.

FIG. 5A shows severe thrombocytopenia following intravenous injection ofIgG immune complexes into CD32A mice (#1) but not following immunecomplex injection into CD32A mice pretreated with effector-deficientMDE-8 IgG1 E269R anti-CD32a mAb (#'s 2-4).

FIG. 5B shows severe thrombocytopenia following intravenous injection ofIgG immune complexes into CD32A mice (#1) but not following immunecomplex injection into CD32A mice pretreated with effector-deficientMDE-8 IgG2 N297A anti-CD32a mAb (#'s 2-3).

FIG. 6A shows the presence of thrombi in the pulmonary blood vessels ofCD32A mice after injection of IgG immune complexes.

FIG. 6B shows no thrombosis in the pulmonary blood vessels following IgGimmune complex injection into CD32A mice pretreated witheffector-deficient MDE-8 IgG1 E269R anti-CD32a mAbs.

FIG. 6C shows the number of pulmonary thrombi in CD32A mice eithertreated with vehicle (#1) or with effector-deficient MDE-8 IgG1 E269Ranti-CD32a mAbs (#'s 2-4).

FIG. 7A shows the presence of thrombi in the pulmonary blood vessels ofCD32A mice after injection of IgG immune complexes.

FIG. 7B shows no thrombosis in the pulmonary blood vessels following IgGimmune complex injection into CD32A mice pretreated witheffector-deficient MDE-8 IgG2 N297A anti-CD32a mAbs.

FIG. 7C shows the number of pulmonary thrombi in CD32A mice eithertreated with vehicle (#1) or with effector-deficient MDE-8 IgG2 N297Aanti-CD32a mAbs (#'s 2-3).

FIG. 8 shows severe thrombocytopenia following intravenous injection ofnative chimeric AT-10 human IgG1 mAb into CD32A mice but not followingintravenous injection of effector-deficient chimeric AT-10 human IgG1E269R.

FIG. 9A shows flow cytometric analysis of whole blood from CD32A miceprior to injection of native chimeric AT-10 human IgG1 mAbs.

FIG. 9B shows flow cytometric analysis of whole blood from CD32A miceafter intravenous injection of native chimeric AT-10 human IgG1 mAbs.

FIG. 9C shows flow cytometric analysis of whole blood from CD32A miceprior to injection of effector-deficient chimeric AT-10 human IgG1 mAbs.

FIG. 9D shows flow cytometric analysis of whole blood from CD32A miceafter the injection of effector-deficient chimeric AT-10 human IgG1mAbs.

FIG. 10 shows severe thrombocytopenia following intravenous injection ofIgG immune complexes into CD32A mice (#1) but not following immunecomplex injection into CD32A mice pretreated with effector-deficientchimeric AT-10 human IgG1 E269R anti-CD32a mAb (#'s 2-3).

FIG. 11A shows flow cytometric analysis of whole blood from CD32A miceafter intravenous injection of IgG immune complexes.

FIG. 11B shows flow cytometric analysis of whole blood fromeffector-deficient chimeric AT-10 human IgG1 E269R anti-CD32a mAbpre-treated CD32A mice after intravenous injection of IgG immunecomplexes.

FIG. 12A shows the presence of thrombi in the pulmonary blood vessels ofCD32A mice after injection of IgG immune complexes.

FIG. 12B shows no thrombosis in the pulmonary blood vessels followingIgG immune complex injection into CD32A mice pretreated witheffector-deficient chimeric AT-10 human IgG1 E269R anti-CD32a mAb.

FIG. 12C shows the number of pulmonary thrombi in CD32A mice eithertreated with vehicle (#1) or with effector-deficient chimeric AT-10human IgG1 E269R anti-CD32a mAb (#'s 2-3).

FIG. 13A shows no drop in body temperature of CD32A mice injected witheffector-deficient humanized AT-10 IgG1 E269R (here and below, this is“hAT-10” mAb).

FIG. 13B shows flow cytometric analysis of whole blood from CD32A micebefore intravenous injection of effector-deficient humanized AT-10 IgG1E269R

FIG. 13C shows flow cytometric analysis of whole blood from CD32A miceafter intravenous injection of effector-deficient humanized AT-10 IgG1E269R.

FIG. 13D shows flow cytometric analysis of whole blood from vehiclepre-treated CD32A mice after intravenous injection of IgG immunecomplexes.

FIG. 13E shows flow cytometric analysis of whole blood fromeffector-deficient humanized AT-10 IgG1 E269R anti-CD32a mAb pre-treatedCD32A mice after intravenous injection of IgG immune complexes.

FIG. 13F shows the number of pulmonary thrombi in CD32A mice eithertreated with vehicle or with effector-deficient humanized AT-10 IgG1E269R anti-CD32a mAb.

FIG. 13G shows the presence of occlusive thrombi in the pulmonary bloodvessels of CD32A mice after injection of IgG immune complexes.

FIG. 13H shows no thrombosis in the pulmonary blood vessels followingIgG immune complex injection into CD32A mice pretreated witheffector-deficient humanized AT-10 IgG1 E269R anti-CD32a mAb.

FIG. 14A shows dose-dependent severe thrombocytopenia followingintravenous injection of native IV.3 human IgG2 mAbs into CD32A mice butnot following intravenous injection of effector-deficient chimeric IV.3human IgG2 N297A mAbs.

FIG. 14B shows no drop in body temperature of CD32A mice injected withnative chimeric IV.3 human IgG2 or with effector-deficient chimeric IV.3human IgG2 N297A.

FIG. 15A shows flow cytometric analysis of whole blood from CD32A miceprior to injection of native chimeric IV.3 human IgG2 mAbs.

FIG. 15B shows flow cytometric analysis of whole blood from CD32A miceafter intravenous injection of native chimeric IV.3 human IgG2 mAbs.

FIG. 15C shows flow cytometric analysis of whole blood from CD32A miceprior to injection of effector-deficient chimeric IV.3 human IgG2 N297AmAbs.

FIG. 15D shows flow cytometric analysis of whole blood from CD32A miceafter intravenous injection of effector-deficient chimeric IV.3 humanIgG2 N297A mAbs.

FIG. 16A shows flow cytometric analysis of whole blood from CD32A miceafter intravenous injection of IgG immune complexes.

FIG. 16B shows flow cytometric analysis of whole blood fromeffector-deficient chimeric IV.3 human IgG2 N297A anti-CD32a mAbpre-treated CD32A mice after intravenous injection of IgG immunecomplexes.

FIG. 17 shows severe thrombocytopenia following intravenous injection ofIgG immune complexes into CD32A mice (#1) but not following immunecomplex injection into CD32A mice pretreated with effector-deficientchimeric IV.3 human IgG2 N297A mAbs (#2-#4).

FIG. 18A shows the presence of occlusive thrombi in the pulmonary bloodvessels of CD32A mice after injection of IgG immune complexes.

FIG. 18B shows no thrombosis in the pulmonary blood vessels followingIgG immune complex injection into CD32A mice pretreated witheffector-deficient chimeric IV.3 human IgG2 N297A anti-CD32a mAbs.

FIG. 18C shows the number of pulmonary thrombi in CD32A mice eithertreated with vehicle (#1) or with effector-deficient chimeric IV.3 IgG2N297A anti-CD32a mAbs (#2-#4).

FIG. 19 shows platelet aggregation response to IgG immune complexes inthe presence of vehicle (#1) or native mouse IV.3 IgG2b (#2) or nativechimeric IV.3 human IgG1 (#3) mAbs.

FIG. 20 shows platelet aggregation response to IgG immune complexes inthe presence of vehicle (#1) or deglycosylated native mouse IV.3 IgG2bmAbs (#2).

FIG. 21 shows platelet aggregation response to IgG immune complexes inthe presence of vehicle (#1) or native chimeric IV.3 human IgG2 mAbs(#'s 2-4).

FIG. 22 shows platelet aggregation response to IgG immune complexes inthe presence of vehicle (#1) or effector-deficient chimeric IV.3 humanIgG2 N297A anti-CD32a mAbs (#'s 2-3).

FIG. 23 shows platelet aggregation response to IgG immune complexes inthe presence of vehicle (#1) or effector-deficient chimeric AT-10 humanIgG1 E269R anti-CD32a mAbs (#2).

FIG. 24 shows platelet aggregation response to IgG immune complexes inthe presence of vehicle (#1) or effector-deficient human MDE-8 IgG1E269R anti-CD32a mAbs (#2). The standard platelet agonist collagen (*)was added to #2 as a positive control in order demonstrate aggregationcompetence of the platelets.

FIG. 25 shows platelet aggregation response to IgG immune complexes inthe presence of vehicle (#1) or effector-deficient humanized IV.3.1 IgG1E269R (here and below, this is “hIV.3.1”) (#2) or native mouse IV.3IgG2b anti-CD32a mAbs (3 nM) (#3).

FIG. 26 shows platelet aggregation response to IgG immune complexes inthe presence of vehicle (#1) or effector-deficient humanized IV.3.1 IgG1E269R (#2) or native mouse IV.3 IgG2b anti-CD32a mAbs (2 nM) (#3). Thestandard platelet agonist collagen (*) was added to #3 as a positivecontrol in order demonstrate aggregation competence of the platelets.

FIG. 27 shows platelet aggregation response to IgG immune complexes inthe presence of vehicle (#1) or effector-deficient humanized IV.3.1 IgG1E269R (6 nM) (#2). The standard platelet agonist collagen (*) was addedto #2 as a positive control in order demonstrate aggregation competenceof the platelets.

FIG. 28 shows platelet aggregation response to IgG immune complexes inthe presence of vehicle (#1) or effector-deficient humanized IV.3.1 IgG1E269R (#2) or native mouse IV.3 IgG2b anti-CD32a mAbs (25 nM) (#3).Buffer (PBS) alone was added as a negative control (#4).

FIG. 29 shows platelet aggregation response to IgG immune complexes inthe presence of vehicle (#1) or effector-deficient humanized IV.3.1 IgG1E269R (40 nM) (#2). The standard platelet agonist collagen (*) was addedto #2 as a positive control in order demonstrate aggregation competenceof the platelets.

FIG. 30 shows platelet aggregation response to IgG immune complexes inthe presence of vehicle (#1) or effector-deficient humanized IV.3.1 IgG1E269R (33 nM) (#2) or effector-deficient human MDE-8 IgG1 E269R (50 nM)(#3).

FIG. 31 shows platelet aggregation response to IgG immune complexes inthe presence of vehicle (#1) or effector-deficient humanized AT-10 IgG1E269R (“hAT-10”; 15 nM) (#2). F(ab′)2 fragments of goatanti-human-F(ab′)2 (#), which lack an Fc-domain, were added to #2 todemonstrate aggregation competence.

FIG. 32 shows platelet aggregation response to IgG immune complexes inthe presence of vehicle (#1) or a combination of effector-deficienthumanized IV.3.1 IgG1 E269R, effector-deficient humanized AT-10 IgG1E269R, and effector-deficient human MDE-8 IgG1 E269R anti-CD32a mAbs(#2). The standard platelet agonist collagen (*) was added to #2 as apositive control in order demonstrate aggregation competence of theplatelets.

FIG. 33 shows a lack of platelet aggregation response to a combinationof effector-deficient chimeric AT-10 human IgG1 E269R,effector-deficient chimeric IV.3 human IgG2 N297A, andeffector-deficient human MDE-8 IgG1 E269R anti-CD32a mAbs.

FIG. 34 shows platelet degranulation in response to IgG antibodies.

FIG. 35 shows platelet degranulation in response to infliximab immunecomplexes (bar 1) and the protective effect of the effector-deficientantibodies described herein (bars 2-4).

FIG. 36A shows flow cytometric analysis of whole blood from CD32A miceprior to injection of a combination of three effector-deficientanti-CD32a mAbs.

FIG. 36B shows flow cytometric analysis of whole blood from CD32A miceafter the injection of a combination of three effector-deficientanti-CD32a mAbs (chimeric AT-10 human IgG1 E269R, chimeric IV.3 humanIgG2 N297A, and human MDE-8 IgG1 E269R; 50 μg of each mAb injected).

FIG. 36C shows flow cytometric analysis of whole blood from CD32A miceprior to injection of a combination of three effector-deficientanti-CD32a mAbs.

FIG. 36D shows flow cytometric analysis of whole blood from CD32A miceafter the injection of a combination of three effector-deficientanti-CD32a mAbs (chimeric AT-10 human IgG1 E269R, chimeric IV.3 humanIgG2 N297A, and human MDE-8 IgG1 E269R; 100 μg of each mAb injected).

FIG. 36E shows no drop in core body temperature of CD32A mice after theinjection of a combination of three effector-deficient anti-CD32a mAbs(chimeric AT-10 human IgG1 E269R, chimeric IV.3 human IgG2 N297A, andhuman MDE-8 IgG1 E269R (denoted “ed-mAbs” in this Figure).

DESCRIPTION OF THE SEQUENCES

Tables 1-5 provide listings of certain sequences referenced herein.

TABLE 1  CDR sequences SEQ ID NO. Description Sequence 1AT-10 VH Kabat CDR1 AA YYWMN 2 AT-10 VH Kabat CDR2 AAEIRLKSNNYATHYAESVKG 3 AT-10 VH Kabat CDR3 AA RDEYYAMDY 4AT-10 VL Kabat CDR1 AA RASESVDNFGISFMN 5 AT-10 VL Kabat CDR2 AA GASNQGS6 AT-10 VL Kabat CDR3 AA QQSKEVPWT 25 IV.3 VH Kabat CDR1 AA NYGMN 26IV.3 VH Kabat CDR2 AA WLNTYTGESIYPDDFKG 27 IV.3 VH Kabat CDR3 AAGDYGYDDPLDY 28 IV.3 VL Kabat CDR1 AA RSSKSLLHTNGNTYLH 29IV.3 VL Kabat CDR2 AA RMSVLAS 30 IV.3 VL Kabat CDR3 AA MQHLEYPLT 54MDE-8 VH Kabat CDR1 AA SYGMH 55 MDE-8 VH Kabat CDR2 AA VIWYDGSNYYYTDSVKG56 MDE-8 VH Kabat CDR3 AA DLGAAASDY 57 MDE-8 VL Kabat CDR1 AARASQGINSALA 58 MDE-8 VL Kabat CDR2 AA DASSLES 59 MDE-8 VL Kabat CDR3 AAQQFNSYPHT 73 hAT-10 VH IMGT CDR1 AA GFTFSYYW 74 hAT-10 VH IMGT CDR2 AAIRLKSNNYAT 75 hAT-10 VH IMGT CDR3 AA NRRDEYYAMDY 76hAT-10 VL IMGT CDR1 AA ESVDNFGISF 77 hAT-10 VL IMGT CDR2 AA GAS 78hAT-10 VL IMGT CDR3 AA QQSKEVPWT 79 hIV.3.1e VH IMGT CDR1 AA GYTFTNYG 80hIV.3.1e VH IMGT CDR2 AA LNTYTGES 81 hIV.3.1e VH IMGT CDR3 AAARGDYGYDDPLDY 82 hIV.3.2b VL IMGT CDR1 AA KSLLHTNGNTY 83hIV.3.2b VL IMGT CDR2 AA RMS 84 hIV.3.2b VL IMGT CDR3 AA MQHLEYPLT 88AT-10 VH IMGT CDR1 AA GFTFSYYW 89 AT-10 VH IMGT CDR2 AA IRLKSNNYAT 90AT-10 VH IMGT CDR3 AA NRRDEYYAMDY 91 AT-10 VL IMGT CDR1 AA ESVDNFGISF 92AT-10 VL IMGT CDR2 AA GAS 93 AT-10 VL IMGT CDR3 AA QQSKEVPWT 94MDE-8 VH IMGT CDR1 AA GFTFSSYG 95 MDE-8 VH IMGT CDR2 AA IWYDGSNY 96MDE-8 VH IMGT CDR3 AA ARDLGAAASDY 97 MDE-8 VL IMGT CDR1 AA QGINSA 98MDE-8 VL IMGT CDR2 AA DAS 99 MDE-8 VL IMGT CDR3 AA QQFNSYPHT 100hIV.3.1c VL IMGT CDR1 AA KSLLHTNGNTY 101 hIV.3.1c VL IMGT CDR2 AA RMS102 hIV.3.1c VL IMGT CDR3 AA MQHLEYPLT

TABLE 2  AT-10 antibody sequences SEQ ID NO. Description Sequence 7AT-10 VH DNA gaagtgaagcttgaggagtctggaggaggcttggtgcaacctggaggatccatgaaactctcctgtgttgcctctggattcactttcagttactactggatgaactgggtccgccagtctccagagaaggggcttgagtgggttgctgaaattagattgaaatctaataattatgcaacacattatgcggagtctgtgaaagggaggttcaccatctcaagagatgattccaaaaataatgtctacctgcaaatgaacaacttaagagctgaagacactggcatttattactgtaacaggcgtgatgagtattacgctatggattattggggtcaagggacgtcggtatctgtgtctagt 8 AT-10 VH AAEVKLEESGGGLVQPGGSMKLSCVASGFTFS YY WMN WVRQSPEKGLEWVA EIRLKSNNYATHYAESVKG RFTISRDDSKNNVYLQMNNLRAEDTGI YYCNR RDEYYAMDY WGQGTSVSVSS 9AT-10 VL DNA gacattgtgctgacccaatctccaggttctttggctgtgtctctagggcagagggccaccatctcctgcagagccagcgaaagtgttgataattttggcattagttttatgaactggttccaacagaaaccaggacagccaccccgactcctcatctatggtgcatccaaccaaggatccggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatgctgcaatgtatttctgtcagcaaagtaaggaggttccgtggacgttcggtggaggcaccaagctggaa atcaaa 10AT-10 VL AA DIVLTQSPGSLAVSLGQRATISC RASESVDNFGIS FMN WFQQKPGQPPRLLIYGASNQGS GVPARFS GSGSGTDFSLNIHPVEEDDAAMYFC QQSKEVP WT FGGGTKLEIK 11hAT-10 VH DNA gaggtgcagctggtggagtctgggggaggcttggtccagcctggagggtccctVariable heavy CDR graftgagactctcctgtgcagcctctggattcaccttctcatactattggatggactgggbased on HV3-72*01 HJ3-01tccgccaggctccagggaaggggctggagtgggttggccgtatcagactgaaa acceptor frameworktctaacaactatgccaccgaatacgccgcgtctgtgaaaggcagattcaccatctcaagagatgattcaaagaactcactgtatctgcaaatgaacagcctgaaaaccgaggacacggccgtgtattactgtaacagaagagatgagtattacgccatggattattggggccaagggacaatggtcaccgtctcttca 12 hAT-10 VH AAEVQLVESGGGLVQPGGSLRLSCAAS GFTFSYYW Variable heavy GDR graftMDWVRQAPGKGLEWVGR IRLKSNNYAT EYAA based on HV3-72*01 HJ3-01SVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYY acceptor framework C NRRDEYYAMDYWGQGTMVTVSS 13 hAT-10 VL DNAgaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagVariable light GDR graftccaccctctcctgcagggccagtgaatctgtggataacttcgggatctccttctta based on KV3-11*01 KJ1*01gcctggtaccaacagaaacctggccaggctcccaggctcctcatctatggagc acceptor frameworkctccaacagggccactggcatcccagccaggttcagtggcagtgggtctggga cagacttcactctcaccatcagcagccragagcctgaagattttgcagtttattac tgtcagcaatctaaagaggtgccatggaccttcggccaagggaccaaggtgga  aatcaaa 14hAT-10 VL AA   EIVLTQSPATLSLSLGERATLSCRAS ESVDNFGISVariable light GDR graft F LAWYQQKPGQAPRLLIY GAS NRATGIPARFSGbased on KV3-11*01 KJ1-01 SGSGTDFTLTISSLEPEDFAVYYC QQSKEVPWT Facceptor framework GQGTKVEIK 15 AT-10 HC IgG1 E269R gaagtgaagcttgaggagtctggaggaggcttggtgcaacctggaggatccat DNA gaaactctcctgtgttgcctctggattcactttcagttactactggatgaactgggt(including constant ccgccagtctccagagaaggggcttgagtgggttgctgaaattagattgaaatct region)aataattatgcaacacattatgcggagtctgtgaaagggaggttcaccatctcaagagatgattccaaaaataatgtctacctgcaaatgaacaacttaagagctgaagacactggcatttattactgtaacaggcgtgatgagtattacgctatggattattggggtcaagggacgtcggtatctgtgtctagtgctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacagagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgg gtaaa 16AT-10 HC IgG1 E269R EVKLEESGGGLVQPGGSMKLSCVASGFTFS YY AA WMNWVRQSPEKGLEWVA EIRLKSNNYATHY (including constant  AESVKGRFTISRDDSKNNVYLQMNNLRAEDTGI region) YYCNR RDEYYAMDY WGQGTSVSVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHRDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK 17AT-10 HC IgG2 N297Agaagtgaagcttgaggagtctggaggaggcttggtgcaacctggaggatccat DNAgaaactctcctgtgttgcctctggattcactttcagttactactggatgaactgggt(including constant ccgccagtctccagagaaggggcttgagtgggttgctgaaattagattgaaatct region)aataattatgcaacacattatgcggagtctgtgaaagggaggttcaccatctcaagagatgattccaaaaataatgtctacctgcaaatgaacaacttaagagctgaagacactggcatttattactgtaacaggcgtgatgagtattacgctatggattattggggtcaagggacgtcggtatctgtgtctagtgctagcaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcgccagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa 18AT-10 HC IgG2 N297A EVKLEESGGGLVQPGGSMKLSCVASGFTFS YY AA WMNWVRQSPEKGLEWVA EIRLKSNNYATHY (including constant  AESVKGRFTISRDDSKNNVYLQMNNLRAEDTGI region) YYCNR RDEYYAMDY WGQGTSVSVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPP CPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREE QFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK 19hAT-10 HC IgG1 E269Rgaggtgcagctggtggagtctgggggaggcttggtccagcctggagggtccct DNAgagactctcctgtgcagcctctggattcaccttctcatactattggatggactggg(including constant tccgccaggctccagggaaggggctggagtgggttggccgtatcagactgaaa region)tctaacaactatgccaccgaatacgccgcgtctgtgaaaggcagattcaccatctcaagagatgattcaaagaactcactgtatctgcaaatgaacagcctgaaaaccgaggacacggccgtgtattactgtaacagaagagatgagtattacgccatggattattggggccaagggacaatggtcaccgtctcttcagctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacagagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctg tctccgggtaaa 20hAT-10 HC IgG1 E269R EVQLVESGGGLVQPGGSLRLSCAAS GFTFSYYW AAMDWVRQAPGKGLEWVGR IRLKSNNYAT EYAA (including constant SVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYY region) C NRRDEYYAMDYWGQGTMVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHRDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 21 AT-10 LC kappa DNAgacattgtgctgacccaatctccaggttctttggctgtgtctctagggcagaggg(including constant ccaccatctcctgcagagccagcgaaagtgttgataattttggcattagttttatg region)aactggttccaacagaaaccaggacagccaccccgactcctcatctatggtgcatccaaccaaggatccggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatgctgcaatgtatttctgtcagcaaagtaaggaggttccgtggacgttcggtggaggcaccaagctggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggag agtgt 22AT-10 LC/ kappa A A DIVLTQSPGSLAVSLGQRATISC RASESVDNFGIS(including constant  FMN WFQQKPGQPPRLLIY GASNQGS GVPARFS region)GSGSGTDFSLNIHPVEEDDAAMYFC QQSKEVP WT FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 23 hAT-10 LC kappa DNAgaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagag(including constant ccaccctctcctgcagggccagtgaatctgtggataacttcgggatctccttctta region)gcctggtaccaacagaaacctggccaggctcccaggctcctcatctatggagcctccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcaatctaaagaggtgccatggaccttcggccaagggaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggga gagtgt 24hAT-10 LC kappa AA  EIVLTQSPATLSLSPGERATLSCRAS ESVDNFGIS(including constant  F LAWYQQKPGQAPRLLIY GAS NRATGIPARFSG region)SGSGTDFTLTISSLEPEDFAVYYC QQSKEVPWT F GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

TABLE 3  IV.3 antibody sequences SEQ ID NO. Description Sequence 31IV.3 VH DNA cagatccagttggtgcagtctggacctgagctgaagaagcctggagagacagtcaagatctcctgcaaggcttctgggtataccttcacaaactatggaatgaactgggtgaagcaggctccaggaaagggtttaaagtggatgggctggttaaacacctacactggagagtcaatatatcctgatgacttcaagggacggtttgccttctcttcggaaacctctgccagcactgcctatttgcagatcaacaacctcaaaaatgaggacatggctacatatttctgtgcaagaggggactatggttacgacgaccctttggactactggggtcaaggaacctcagtcaccgtctcctca 32 IV.3 VH AAQIQLVQSGPELKKPGETVKISCKASGYTFT NYG MN WVKQAPGKGLKWMG WLNTYTGESIYPD DFKGRFAFSSETSASTAYLQINNLKNEDMATYF CAR GDYGYDDPLDY WGQGTSVTVSS 33 IV.3 VL DNAgacattgtgatgacccaggctgcaccctctgtacctgtcactcctggagagtcagtatccatctcctgcaggtctagtaagagtctcctgcatactaatggcaacacttacttgcattggttcctacagaggccaggccagtctcctcagctcctgatatatcggatgtccgtccttgcctcaggagtcccagacaggttcagtggcagtgggtcaggaactgctttcacactgagcatcagtagagtggaggctgaggatgtgggtgttttttactgtatgcaacatctagaatatccgctcacgttcggtgctgggaccaagctggaac tgaaa 34IV.3 VL AA DIVMTQAAPSVPVTPGESVSISC RSSKSLLHTNG NTYLH WFLQRPGQSPQLLIYRMSVLAS GVPDR FSGSGSGTAFTLSISRVEAEDVGVFYC MQHLEY PLT FGAGTKLELK 35hIV.3.1e VH DNA caggtgcagctggtgcaatctgggtctgagttgaagaagcctggggcctcagtgVariable heavy CDR graft aaggtttcctgcaaggcttctggatacaccttcactaactatggtatgaattgggtbased on HV7-4-1*2 gcgacaggcccctggacaagggcttgagtggatgggatggctcaacacctacaHJ6*01 acceptor ctggggagtcaacgtatgcccagggcttcacaggacggtttgtcttctccttggaframework cacctctgtcagcacggcatatctgcagatcagcagcctaaaggctgaggacactgccgtgtattactgtgcgagaggggactatggttacgacgaccctttggactactgggggcaagggaccacggtcaccgtctcctca 36 hIV.3.1e VH AAQVQLVQSGSELKKPGASVKVSCKAS GYTFTNY Variable heavy CDR graft GMNWVRQAPGQGLEWMGW LNTYTGES TYA based on HV7-4-1*2QGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYY HJ6*01 acceptor C ARGDYGYDDPLDYWGQGTTVTVSS framework 37 hIV.3.2d VH DNAcaggtgcagctggtgcagtctggccatgaggtgaagcagcctggggcctcagtVariable heavy CDR graftgaaggtctcctgcaaggcttctgggtataccttcacaaactatggaatgaactggbased on HV7-81*01 gtgaaacaggcccctggacaagggcttaagtggatgggctggttaaacacctaHJ6*01 acceptor cactggagagtcaatatatcctgatgacttcaagggacggtttgccttctccagtframework gacacctctgccagcacagcatacctgcagatcaacaacctaaaggctgaggacatggccatgtatttctgtgcgagaggggactatggttacgacgaccctttggactactgggggcaagggaccacggtcaccgtctcctca 38 hIV.3.2d VH AAQVQLVQSGHEVKQPGASVKVSCKASGYTFT NY Variable heavy CDR graft GMNWVKQAPGQGLKWMG WLNTYTGE SIYP based on HV7-81*01 DDFKGRFAFSSDTSASTAYLQINNLKAEDMAMY HJ6*01 acceptor FCAR GDYGYDDPLDYWGQGTTVTVSS framework 39 hIV.3.1c VL DNAgatattgtgatgacccagactccactctccctgcccgtcacccctggagagccgVariable light CDR graftgcctccatctcctgcaggtctagtaagtctctgctgcataccaacgggaacacctbased on KV2-40*01atttggactggtacctgcagaagccagggcagtctccacagctcctgatctatag KJ4*02 acceptorgatgtcctatcgggcctctggagtcccagacaggttcagtggcagtgggtcagg frameworkcactgatttcacactgaaaatcagcagggtggaggctgaggatgttggagtttattactgcatgcagcatctggagtatccactgaccttcggcggagggaccaaggtg gagatcaaa 85hIV.3.1c VL AA DIVMTQTPLSLPVTPGEPASISCRSS KSLLHTNGVariable light CDR graft NTY LDWYLQKPGQSPQLLIY RMS YRASGVPDRbased on KV2-40*01 FSGSGSGTDFTLKISRVEAEDVGVYYC MQHLE KJ4*02 acceptorYPLT FGGGTKVEIK framework 40 hIV.3.2b VL DNAgatattgtgatgacccagactccactctccctgcccgtcacccctggagagccgVariable light CDR graftgcctccatctcctgcaggtctagtaagagtctcctgcatactaatggcaacacttbased on KV2-40*01acttgcattggtacctgcagaagccagggcagtctccacagctcctgatatatcg KJ4*02 acceptorgatgtccgtccttgcctcaggagtcccagacaggttcagtggcagtgggtcagg frameworkcactgatttcacactgaaaatcagcagggtggaggctgaggatgttggagtttattactgcatgcaacatctagaatatccgctcacgttcggcggagggaccaaggtg gagatcaaa 41hIV.3.2b VL AA DIVMTQTPLSLPVTPGEPASISCRSS KSLLHTNGVariable light CDR graft NTY LHWYLQKPGQSPQLLIY RMS VLASGVPDRbased on KV2-40*01 FSGSGSGTDFTLKISRVEAEDVGVYYC MQHLE KJ4*02 acceptorYPLT FGGGTKVEIK framework 42 IV.3 HC IgG1 E269Rcagatccagttggtgcagtctggacctgagctgaagaagcctggagagacagt DNAcaagatctcctgcaaggcttctgggtataccttcacaaactatggaatgaactgg(including constant gtgaagcaggctccaggaaagggtttaaagtggatgggctggttaaacacctac region)actggagagtcaatatatcctgatgacttcaagggacggtttgccttctcttcggaaacctctgccagcactgcctatttgcagatcaacaacctcaaaaatgaggacatggctacatatttctgtgcaagaggggactatggttacgcgaccctttggactactggggtcaaggaacctcagtcaccgtctcctcagctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacagagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctg tctccgggtaaa 43IV.3 HCIgGl E269RAA QIQLVQSGPELKKPGETVKISCKASGYTFT NYG(including constant  MN WVKQAPGKGLKWMG WLNTYTGESIYPD region) DFKGRFAFSSETSASTAYLQINNLKNEDMATYF CAR GDYGYDDPLDY WGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHRDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK 44IV.3 HGIgG2 N297A DNAcagatccagttggtgcagtctggacctgagctgaagaagcctggagagacagt(including constant caagatctcctgcaaggcttctgggtataccttcacaaactatggaatgaactgg region)gtgaagcaggctccaggaaagggtttaaagtggatgggctggttaaacacctacactggagagtcaatatatcctgatgacttcaagggacggtttgccttctcttcggaaacctctgccagcactgcctatttgcagatcaacaacctcaaaatgaggacatggctacatatttctgtgcaagaggggactatggttacgacgaccctttggactactggggtcaaggaacctcagtcaccgtctcctcagctagcaccaagggcccatcggtcttcccctggcgccctgctccaggagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcgccagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa 45IV.3 HC IgG2 N297A AA QIQLVQSGPELKKPGETVKISCKASGYTFT NYG(including constant  MN WVKQAPGKGLKWMG WLNTYTGESIYPD region) DFKGRFAFSSETSASTAYLQINNLKNEDMATYF CAR GDYGYDDPLDY WGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVNHKPSNTKVDKKVERKSCCVECPP CPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREE QFASTYRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK 46hIV.3.1e HC IgG1 E269Rcaggtgcagctggtgcaatctgggtctgagttgaagaagcctggggcctcagtg DNAaaggtttcctgcaaggcttctggatacaccttcactaactatggtatgaattgggt(including constant gcgacaggcccctggacaagggcttgagtggatgggatggctcaacacctaca region)ctggggagtcaacgtatggcccagggcttcacaggacggtttgtcttctccttggacacctctgtcagcacggcatatctgcagatcagcagcctaaaggctgaggacactgccgtgtattactgtgcgagaggggactatggttacgacgaccctttggactactgggggcaagggaccacggtcaccgtctcctcagctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacagagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtggccacagagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctg tctccgggtaaa 47hIV.3.1e HC IgG1 E269R QVQLVQSGHEVKQPGETVKISCKAS GYTFTNY AA GMNWVKQAPGKGLKWMGW LNTYTGES TYA (including constant DFKGRFAFSSDTSASTAYLQINNLKNEDMAMYF region) C ARGDYGYDDPLDYWGQGTTVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHRDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 48 hIV.3.2d HC IgG1 E269Rcaggtgcagctggtgcagtctggccatgaggtgaagcagcctggggcctcagt DNAgaaggtctcctgcaaggcttctgggtataccttcacaaactatggaatgaactgg(including constant gtgaaacaggcccctggacaagggcttaagtggatgggctggttaaacaccta region)cactggagagtcaatatatcctgatgacttcaagggacggtttgccttctccagtgacacctctgccagcacagcatacctgcagatcaacaacctaaaggctgaggacatggccatgtatttctgtgcgagaggggactatggttacgacgaccctttggactactgggggcaagggaccacggtcaccgtctcctcagctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacagagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccagtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccct gtctccgggtaaa 49hIV.3.1d HC IgG1 E269R QVQLVQSGHEVKQPGETVKISCKAS GYTFTNY AA GMNWVKQAPGKGLKWMGW LNTYTGES IYPD (including constant DFKGRFAFSSDTSASTAYLQINNLKNEDMAMYF region) C ARGDYGYDDPLDYWGQGTTVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHRDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 50 IV.3 LC kappa DNAgacattgtgatgacccaggctgcaccctctgtacctgtcactcctggagagtcag(including constant tatccatctcctgcaggtctagtaagagtctcctgcatactaatggcaacacttac region)ttgcattggttcctacagaggccaggccagtctcctcagctcctgatatatcggatgtccgtccttgcctcaggagtcccagacaggttcagtggcagtgggtcaggaactgctttcacactgagcatcagtagagtggaggctgaggatgtgggtgttttttactgtatgcaacatctagaatatccgctcacgttcggtgctgggaccaagctggaactgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggaga gtgt 51IV.3 LC kappa AA DIVLTQSPGSLAVSLGEPASISCRR KSLLHTING(including constant  NTY LHWLQKPGQSPRLLIY RM S VLASVPDR region)FSGSGTDFTLKISRVEAEDVGVYYC M Q HLE YPL T FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 86 hIV.31c LC kappa DNAgatattgtgatgacccagactccactctccctgcccgtcacccctggagagccg(including constant gcctccatctcctgcaggtctagtaagtctctgctgcataccaacgggaacacct region)atttggactggtacctgcagaagccagggcagtctccacagctcctgatctataggatgtcctatcgggcctctggagtcccagacaggttcagtggcagtgggtcaggcactgatttcacactgaaaatcagcagggtggaggctgaggatgttggagtttattactgcatgcagcatctggagtatccactgaccttcggcggagggaccaaggtggagatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggg gagagtgt 87hIV.31c LC kappa AA DIVLTQSPGSLAVSLGEPASISCRR KSLLHTING(including constant  NTY LHWLQKPGQSPRLLIY RM S VLASVPDR region)FSGSGTDFTLKISRVEAEDVGVYYC M Q HLE YPL T FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 52 hIV.3.2b LC kappa DNAgatattgtgatgacccagactccactctccctgcccgtcacccctggagagccg(including constant gcctccatctcctgcaggtctagtaagtctctgctgcataccaacgggaacacct region)atttggactggtacctgcagaagccagggcagtctccacagctcctgatctataggatgtcctatcgggcctctggagtcccagacaggttcagtggcagtgggtcaggcactgatttcacactgaaaatcagcagggtggaggctgaggatgttggagtttattactgcatgcagcatctggagtatccactgaccttcggcggagggaccaaggtggagatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggg gagagtgt 53hIV.3.2b LC kappa AA DIVLTQSPGSLAVSLGEPASISCRR KSLLHTING(including constant  NTY LHWLQKPGQSPRLLIY RM S VLASVPDR region)FSGSGTDFTLKISRVEAEDVGVYYC M Q HLE YPL T FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

TABLE 4  MDE-8 antibody sequences SEQ ID NO. Description Sequence 60MDE-8 VH DNA caggcacctggtggagtctgggggaggcgtggtccagcctgggaggtccct gagactctcctgtgcagcgtctggattcaccttcagtagctatggcatgcactgggtccgccaggc tccaggcaaggggctggagtgggtggcagttatatggtatgat ggaagtaattactactatacagnctccgtgaagggccgattcaccatctccagag acaartccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggac acggctgtgtattactgtgcgagagatctgggggcagcagcttctgactactgg ggccagggaaccctggtcaccgtctcctca 61 MDE-8 VH AAQVHLVESGGGVVPGRSLRLSCAASGFTFSSYG MH WVRQAPGKGLEWVA VIWYDGSNYYYTDS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARD LGAAASDY WGQGTLVTVSS 62 MDE-8 VL DNAgccatccagttgacccagtctccatcctccctgtctgcatagtaggagacagag tcaccatcacttgccgggcaagtcagggcattaacagtgctttagcctggtatca gcagaaaccagggaaagctcctaagctcctgatctargatgcctccagtttgga aagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgtcaacagttta ataggttaccctcatacttttggccaggggnccaagctggagatcaaa 63 MDE-8 VL AAAIQLTQSPSSLSASVGDRTVTITC RASQGINSALA WYQQKPGKAPKLLIY DASSLES GVPSRRSGSGSGTDFTLTISSLQPEDFATYYC QQFNSYPHT FGQ GTKLEIK 64 MDE-8 HC IgGl E269R DNAcaggtgcacctggtggagtctgggggaggcgtggtccagcctgggaggtccct(including constant region)gagactctcctgtgcagcgtctggattcaccttcagtagctatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgatggaagtaattactactatacagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagatctgggggcagcagcttctgactactggggccagggaaccctggtcaccgtctcctcagctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctgggggaccgtcagtcttcctcttccccccaaaacccaccggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacagagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc cgggtaaa 65MDE-8 HC IgG1 E269R AA  QVHLVESGGGVVPGRSLRLSCAASGFTFS SYG(including constant region) MH WVRQAPGKGLEWVA VIWYDGSNYYYTDS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARD LGAAASDY WGQGTLVTVSSASTKGPSVFPLAPSSLSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHRDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK 66MDE-8 HC IgG2 N297A DNA caggtgcacctggtggagtctgggggaggcgtggtccagcctgggaggtccct(including constant region)gagactctcctgtgcagcgtctggattcaccttcagtagctatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgatggaagtaattactactatacagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagatctgggggcagcagcttctgactactggggccagggaaccctggtcaccgtctcctcagctagcaccaagggcccatcggtcttccccctggcgcctgctccaggagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcgccagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa 67MDE-8 HC IgG2 N297A AA QVHLVESGGGVVPGRSLRLSCAASGFTFS SYG(including constant region) MH WVRQAPGKGLEWVA VIWYDGSNYYYTDS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC AR D LGAAASDY WGQGTLVTVSSASTKGPSVFPLAPSSLSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQFAS TFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 68MDE-8 LC kappa DNAgccatccagttgacccagtctccatcctccctgtctgcatctgtaggagacagag(including constant region)tcaccatcacttgccgggcaagtcagggcattaacagtgctttagcctggtatcagcagaaaccagggaaagctcctaagctcctgatctatgatgcctccagtttggaaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgtcaacagtttaatagttaccctcatacttttggccaggggaccaagctggagatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt 69 MDE-8 LC kappa AAAIQLTQSPSSLSASVGDRTVTITC RASQGINSALA (including constant region)WYQQKPGKAPKLLIY DASSLES GVPSRRSGSGS GTDFTLTISSLQPEDFATYYC QQFNSYPHT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC

TABLE 5  other sequences SEQ ID NO. Description Sequence 70NP_001129691.1 MTMETQMSQNVCPRNLWLLQPLTVLLLL CD32a-H131 ASADSQAAAPPKAVLKLEPPWINVLQED [Homo sapiens] SVTLTCQGARSPESDSIQWFHNGNLIPTHTQPSYRFKANNNDSGEYTCQTGQTSLS DPVHLTVLSEWLVLQTPHLEFQEGETIMLRCHSWKDKPLVKVTFFQNGKSQKFS H L DPTFSIPQANHSHSGDYHCTGNIGYTLFSSKPVTITVQVPSMGSSSPMGIIVAVVI ATAVAAIVAAVVALIYCRKKRISANSTDPVKAAQFEPPGRQMIAIRKRQLEETNND YETADGGYMTLNPRAPTDDDKNIYLTLP PNDHVNSNN 71NP_001129691.1: MTMETQMSQNVCPRNLWLLQPLTVLLLL p.His167ASADSQAAAPPKAVLKLEPPWINVLQED Arg CD32a-R131 SVTLTCQGARSPESDSIQWFHNGNLIPT [Homo sapiens] HTQPSYRFKANNNDSGEYTCQTGQTSLSDPVHLTVLSEWLVLQTPHLEFQEGETIM LRCHSWKDKPLVKVTFFQNGKSQKFS R LDPTFSIPQANHSHSGDYHCTGNIGYTLF SSKPVTITVQVPSMGSSSPMGIIVAVVIATAVAAIVAAVVALIYCRKKRISANSTD PVKAAQFEPPGRQMIAIRKRQLEETNNDYETADGGYMTLNPRAPTDDDKNIYLTLP PNDHVNSNN 72 NP_003992.3 MGILSFLPVLATESDWADCKSPQPWGHM CD32b isoform 1LLWTAVLFLAPVAGTPAAPPKAVLKLEP [Homo sapiens] QWINVLQEDSVTLTCRGTHSPESDSIQWFHNGNLIPTHTQPSYRFKANNNDSGEYT CQTGQTSLSDPVHLTVLSEWLVLQTPHLEFQEGETIVLRCHSWKDKPLVKVTFFQN GKSKKFSRSDPNFSIPQANHSHSGDYHCTGNIGYTLYSSKPVTITVQAPSSSPMGI IVAVVTGIAVAAIVAAVVALIYCRKKRISALPGYPECREMGETLPEKPANPTNPDE ADKVGAENTITYSLLMHPDALEEPDDQN RI

DESCRIPTION OF THE EMBODIMENTS

In one embodiment, a method for treating a CD32a-mediated disease ordisorder in a human subject is encompassed, wherein a therapeuticallyeffective amount of one or more effector-deficient anti-CD32a monoclonalantibodies as described herein, is administered to a human subject,thereby treating the CD32a-mediated disease or disorder.

In one embodiment, the anti-CD32a monoclonal antibody is capable of 1)preventing activation of CD32a by IgG immune complexes; and 2) has an Fcregion that has been altered so as to reduce or eliminate Fc-binding toCD16, CD32, or CD64 type IgG receptors.

In one embodiment, the anti-CD32a monoclonal antibody is capable of 1)preventing activation of CD32a by IgG immune complexes; and 2) has an Fcregion that has been altered so as to reduce or eliminate Fc-binding toCD16, CD32, and CD64 type IgG receptors.

In one embodiment, the reduction in Fc-binding to CD16, CD32, and/orCD64 is a complete reduction as compared to an effector-competentantibody control. In other aspects, the reduction in about 50%, about60%, about 70%, about 80%, about 90%, or about 95%, or more, as comparedto an effector-competent antibody control.

Antibodies

Any effector-deficient anti-CD32a antibody may be used in the methodembodiments. The antibodies of the composition and method embodimentscomprise at least a portion of the Fc region.

In one embodiment, the effector-deficient antibody is an AT-10, IV.3, orMDE-8 antibody comprising one or more of the CDRs described for eachantibody, respectively, as in Tables 1-5, and is effector-deficient. Inother embodiments, the effector-deficient antibody is an AT-10, IV.3, orMDE-8 antibody comprising the variable heavy and light chains describedfor each antibody, respectively, as in Tables 1-5, and iseffector-deficient. In other embodiments, the effector-deficientantibody is an AT-10, IV.3, or MDE-8 antibody comprising the full-lengthheavy and full-length light chains described for each antibody,respectively, as in Tables 1-5, and is effector-deficient.

In one embodiment, the effector-deficient antibody is an AT-10, IV.3, orMDE-8 antibody comprising one or more of the CDRs of that antibody,wherein the CDRs are identical to the CDR sequences described for eachantibody, respectively, in Tables 1-5, or wherein one, two, or three ofthe CDRs have 1 or 2 mutations as compared to the sequences describedfor each antibody, as in Table 1, and is effector-deficient.

In other embodiments, the effector-deficient antibody is an AT-10, IV.3,or MDE-8 antibody comprising the variable heavy and light chainsdescribed in Tables 1-5 for each antibody, respectively, wherein thevariable heavy and light chains are 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to the variable heavy and variable light chainsdescribed in Tables 1-5 for each antibody, respectively, and wherein theantibody is effector-deficient.

In other embodiments, the effector-deficient antibody is an AT-10, IV.3,or MDE-8 antibody comprising a full length heavy and light chaindescribed in Tables 1-5 for each antibody, respectively, or a variableheavy and light chain that is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99° A identical to a heavy and light chain described in Tables 1-5 foreach antibody, respectively, and is effector-deficient.

The antibody compositions of the invention, as well as the antibodiesused in the methods and uses described herein, are capable of preventingactivation of CD32a by IgG immune complexes. Whether an antibody iscapable of preventing activation of CD32a by IgG immune complexes can betested by methods well known in the art, namely, by testing washedplatelets for aggregation or degranulation responses to IgG immunecomplexes, as per the “IgG Immune Complex Test” described below. See,e.g., Meyer T et al. Bevacizumab immune complexes activate platelets andinduce thrombosis in FCGR2A transgenic mice (January 2009) J ThrombHaemost 7:171; PubMed ID: 18983497.

“IgG Immune Complex Test”: The following steps can determine whether anantibody can prevent activation of CD32a by IgG immune complexes. First,for example, human platelets can be isolated from other blood cells by“washing” methods (see, e.g., Meyer T et al. (January 2009) J ThrombHaemost 7:171; PubMed ID: 18983497). Alternatively, platelets fromFCGR2A transgenic mice can be isolated using similar methods (see, e.g.,Robles-Carrillo L et al. Anti-CD40L immune complexes potently activateplatelets in vitro and cause thrombosis in FCGR2A transgenic mice(August 2010) J Immunol 185:1577; PubMed ID: 20585032). Second, suchwashed platelets can then be used to test for CD32a-mediated activationby IgG antibodies known to activate human CD32a, for example anti-CD9mAb (e.g., as in PubMed ID: 18983497, op cit), or anti-CD40L mAb, M90(e.g., as in PubMed ID: 20585032, op di). In order to activate CD32a onwashed platelets, some antibodies may need to be clustered by antigen soas to form an immune complex (IC), as is the case for M90, which iscombined with CD40L prior to exposure to washed platelets.CD32a-activating antibodies can be identified using a plateletaggregometer, such as a Chrono-Log model 490 series aggregometer. If theantibody causes platelet aggregation after introduction into theaggregometer cuvette, and such aggregation is prevented by an anti-CD32ablocking antibody (e.g., such as IV.3, AT-10, or MDE-8; many others arecommercially available and are known to those skilled in the art), thenthe antibody specifically activates platelet CD32a and is thereforesufficient for use as a required reagent in the “IgG Immune ComplexTest”. An alternative to the washed platelet aggregation test is theserotonin release assay (or “SRA”), which measures plateletdegranulation (see, e.g., PubMed IDs 18983497 and 20585032 op cit).CD32a is the only IgG receptor on human platelets; therefore, thesetests are capable of specifically identifying CD32a-activatingantibodies. The third step in the “IgG Immune Complex Test” requiresexposure of washed platelets to candidate anti-CD32a antibodies prior tointroduction of the CD32a-activating IgG antibody. For example, washedhuman platelets suspended in assay buffer (typically, 250/nanoliter) areplaced in an aggregometer cuvette. The instrument settings are adjustedso as to establish an assay signal range and baseline. Next, thecandidate anti-CD32a blocking antibody (e.g., IV.3, AT-10, or MDE-8) isintroduced into the cuvette (typically at or near 10 micrograms permilliliter). Next, the platelet activating IgG antibody or IgG immunecomplex (e.g., M90+CD40L, typically at 50-500 nM final assayconcentration) is added to the platelet suspension in the cuvette.Finally, platelet aggregation is monitored for at least one minute (or,typically, more than five minutes) to assess whether the anti-CD32a mAbprevents IgG antibody/immune complex-induced platelet aggregation. If ananti-CD32a antibody, using these steps, can prevent the activation ofCD32a by IgG antibodies or IgG immune complexes, as evidenced byinhibition of aggregation (or degranulation if the SRA is used), thenthe anti-CD32a antibody satisfies the “IgG Immune Complex Test”.Similarly, if an anti-CD32a antibody lacks the capacity to preventplatelet aggregation and degranulation, said anti-CD32a antibody failsto satisfy the “IgG Immune Complex Test”.

In the Examples included herein, FIGS. 19-33 demonstrate by washedplatelet aggregation (i.e., using the “IgG Immune Complex Test”) thatmouse IV.3, chimeric IV.3, and humanized IV.3, that chimeric AT-10 andhumanized AT-10, and that human MDE-8 IgG anti-CD32a mAbs all satisfythe “IgG Immune Complex Test”, regardless of whether such anti-CD32aantibodies are of the IgG1 or IgG2 isotype subclass, and regardless ofwhether such anti-CD32a mAbs have native or effector-deficient Fcregions. As an alternative to the use of platelet aggregation as an “IgGImmune Complex Test”, FIGS. 34 and 35 demonstrate similar results forIV.3, AT-10, and MDE-8 antibody variants using the SRA instead ofplatelet aggregation; here also, all tested antibodies preventIC-induced platelet activation and therefore satisfy the “IgG ImmuneComplex Test”.

a. Effector-Deficiency

The antibody compositions of the invention, as well as the antibodiesused in the methods and uses described herein, are “effector-deficient.”As used herein, an “effector-deficient” antibody is defined as anantibody having an Fc region that has been altered so as to reduce oreliminate Fc-binding to CD16, CD32, and/or CD64 type IgG receptors.

In one embodiment, the reduction in Fc-binding to CD16, CD32, and/orCD64 is a complete reduction as compared to an effector-competentcontrol. In other aspects, the reduction in about 50%, about 60%, about70%, about 80%, about 90%, or about 95%, or more, as compared to aneffector-competent antibody control. Methods for determining whether anantibody has a reduced Fc-binding to CD16, CD32, and/or CD64 are wellknown in the art. See, e.g., US20110212087 A1, WO 2013165690, and VafaO. et al. An engineered Fc variant of an IgG eliminates all immuneeffector functions via structural perturbations (January 2014) Methods65:114; PubMed ID: 23872058.

In further embodiments, an effector-deficient anti-CD32a antibody is anantibody that is capable of 1) preventing activation of CD32a by IgGimmune complexes; 2) has an Fc region that has been altered so as toreduce or eliminate Fc-binding to CD16, CD32, and/or CD64 type IgGreceptors; and 3) does not induce Fc-mediated adverse host reactionsfollowing administration.

Whether the anti-CD32a effector-deficient antibodies of the presentinvention are capable of inducing an adverse host reaction followingadministration can be tested by the “Immobilized IgG Test” describedbelow.

“Immobilized IgG Test”: The following steps can determine whether ananti-CD32a antibody is capable of inducing an IgG-mediated adversereaction following intravenous administration into a host animal. Thehost animal must be a mammal and must display CD32 IgG receptors havingat least one epitope to which the anti-CD32a antibody to be tested isknown to bind as an antigen. For example, IV.3 is an IgG mAb known tobind CD32a antigen (e.g., as in SEQ ID NO: 70, and as in SEQ ID NO: 71;see, e.g., Rosenfeld S I et al. Human platelet Fc receptor forimmunoglobulin G. Identification as a 40,000-molecular-weight membraneprotein shared by monocytes (December 1985) J Clin Invest 76:2317;PubMed ID: 2934409); AT-10 is an IgG mAb known to bind CD32 antigen(e.g., as in SEQ ID NOs: 70-72; see e.g., Greenman J et al.Characterization of a new monoclonal anti-Fc gamma RII antibody, AT10,and its incorporation into a bispecific F(ab′)2 derivative forrecruitment of cytotoxic effectors (November 1991) Mol Immunol 28:1243;PubMed ID: 1835758); and MDE-8 is an IgG mAb known to bind CD32 antigen(e.g., as in SEQ ID NOs: 70-72; see e.g., van Royen-Kerkhof A et al. Anovel human CD32 mAb blocks experimental immune haemolytic anaemia inFcgammaRIIA transgenic mice (July 2005) Br J Haematol 130:130; PubMedID: 15982355). One suitable host animal for use in the “Immobilized IgGTest” for anti-CD32a mAbs is the FCGR2A mouse (“B6;SJL-Tg(FCGR2A)11Mkz/J” mice, #003542, The Jackson Laboratory, Bar Harbor, Me., USA).Other suitable CD32-positive host animals are known to those skilled inthe art. The “Immobilized IgG Test” is then conducted by, for example,injecting the purified anti-CD32a test antibody (preferably inphysiologic saline, phosphate buffered saline, or another suitably inertvehicle) into the tail vein of (in this case) the FCGR2A (i.e., CD32A)mouse. Typically, 50-100 micrograms is injected; however, lack ofreaction may suggest greater quantities of antibody should be injected:for example, 120 micrograms or 140 micrograms may be required to elicita reaction. Quantities greater than 150 micrograms are typically notrequired for FCGR2A mice. Immediately following injection of the testantibody (in this example, the anti-CD32a mAb), the animal in monitoredfor core body temperature (typically, using a rectal thermometer) every10 minutes for at least 20 minutes post injection (baseline temperatureis established prior to test mAb injection). A temperature drop of morethan two degrees celcius (i.e., hypothermia) that is sustained for morethan five minutes, represents an adverse reaction indicating that theanti-CD32a test mAb failed to satisfy the “Immobilized IgG Test”.Additionally, at least twenty minutes after injection of the anti-CD32atest mAb, and preferably thirty minutes after injection of theanti-CD32a test mAb, whole blood is collected from the host animal(retro-orbitally, or by venipuncture) and analyzed to assess changes inthe number of circulating target cells. Cell counts can be obtained byflow cytometry, by automated cell counter, or by use of a hemocytometer.In the case of testing anti-CD32a mAbs in FCGR2A mice, baseline plateletcounts are obtained on the day prior to testing, or at least one tothree hours prior to injection of the anti-CD32a test mAb. Note that theprocess of blood draw, and in particular serial blood draws, can reduceapparent cell counts. Typically, baseline platelet counts in FCGR2A micewill exceed 700 per nanoliter, and are more typically greater than 800per nanoliter, and may be as high as 1200, 1500, 1800, or 2000 pernanoliter. In the case of testing anti-CD32a mAbs in FCGR2A (CD32A)mice, a drop in circulating platelet counts of greater than 50%represents an adverse reaction indicating that the anti-CD32a test mAbfailed to satisfy the “Immobilized IgG Test”. In contrast, if 50 or moremicrograms of an anti-CD32a mAb is intravenously injected into CD32Amice and core body temperature does not drop more than two degreescelcius for more than five minutes and circulating platelet counts arenot reduced by more than 50% within thirty minutes, the anti-CD32aantibody satisfies the “Immobilized IgG Test”.

In the Examples included herein, FIGS. 1-17 and FIG. 36 demonstrate(i.e., using the “Immobilized IgG Test”) that effector-deficient but notnative formats of chimeric IV.3, chimeric AT-10, humanized AT-10, andhuman MDE-8 IgG anti-CD32a mAbs satisfy the “Immobilized IgG Test”,regardless of whether such anti-CD32a antibodies are of the IgG1 or IgG2isotype subclass. Notably, all native anti-CD32a IgG mAbs tested by the“Immobilized IgG Test” failed to satisfy the “Immobilized IgG Test” inthese examples, while all effector-deficient anti-CD32a IgG mAbs testedby the “Immobilized IgG Test” satisfied the “Immobilized IgG Test”.

Methods for engineering effector-deficient antibodies with reducedcapacity for Fc-dependent binding to CD16, CD32, and/or CD64 are wellknown in the art. For example, in order to achieve this result, aneffector-deficient antibody may have one or more of the followingmutations: E233P, G237M, D265A, D265N, E269R, D270A, D270N, N297A,N297Q, N297D, N297R, S298N, T299A (numbering is EU index of Kabat).

In certain embodiments, the Fc region mutation is selected fromM252Y+S254T+T256E, G385D+Q386P+N389S, and H433K+N434F+Y436H, which aremutations known to extend circulating half-life of the therapeuticantibody (see, e.g., U.S. Pat. No. 8,323,962).

In certain embodiments, the anti-CD32a mAbs of the invention aremodified to remove T-cell epitopes, which are known in the art topromote immunogenicity.

1. Effector-Deficient AT-10 Monoclonal Antibodies

In one embodiment, the effector-deficient anti-CD32a antibody is aneffector-deficient AT-10 antibody. In one aspect, the AT-10 antibodycomprises:

-   -   a. a heavy chain variable region CDR1 sequence comprising a        sequence that is identical to the sequence YYWMN (SEQ ID NO: 1)        or GFTFSYYW (SEQ ID NO: 73 and SEQ ID NO: 88), or is a sequence        having 1 amino acid difference as compared to YYWMN (SEQ ID        NO: 1) or GFTFSYYW (SEQ ID NO: 73 and SEQ ID NO: 88);    -   b. a heavy chain variable region CDR2 sequence comprising a        sequence that is identical to the sequence EIRLKSNNYATHYAESVKG        (SEQ ID NO: 2) or IRLKSNNYAT (SEQ ID NO: 74 and SEQ ID NO: 89),        or is a sequence having 1 or 2 amino acid differences as        compared to EIRLKSNNYATHYAESVKG (SEQ ID NO: 2) or IRLKSNNYAT        (SEQ ID NO: 74 and SEQ ID NO: 89);    -   c. a heavy chain variable region CDR3 sequence comprising a        sequence that is at identical to the sequence RDEYYAMDY (SEQ ID        NO: 3) or NRRDEYYAMDY (SEQ ID NO: 75 and SEQ ID NO: 90), or is a        sequence having 1 or 2 amino acid differences as compared to        RDEYYAMDY (SEQ ID NO: 3) or NRRDEYYAMDY (SEQ ID NO: 75 and SEQ        ID NO: 90);    -   d. a light chain variable region CDR1 sequence comprising a        sequence that is at identical to the sequence RASESVDNFGISFMN        (SEQ ID NO: 4) or ESVDNFGISF (SEQ ID NO: 76 and SEQ ID NO: 91),        or is a sequence having 1 or 2 amino acid differences as        compared to RASESVDNFGISFMN (SEQ ID NO: 4) or ESVDNFGISF (SEQ ID        NO: 76 and SEQ ID NO: 91;    -   e. a light chain variable region CDR2 sequence comprising a        sequence that is at identical to the sequence GASNQGS (SEQ ID        NO: 5) or GAS (SEQ ID NO: 77 and SEQ ID NO: 92), or is a        sequence having 1 or 2 amino acid differences as compared to        GASNQGS (SEQ ID NO: 5) or GAS (SEQ ID NO: 77 and SEQ ID NO: 92);        and    -   f. a light chain variable region CDR3 sequence comprising a        sequence that is identical to the sequence QQSKEVPWT (SEQ ID        NO: 6) or QQSKEVPWT (SEQ ID NO:78 and SEQ ID NO: 93), or is a        sequence having 1 or 2 amino acid differences as compared to        QQSKEVPWT (SEQ ID NO: 6) or QQSKEVPWT (SEQ ID NO:78 and SEQ ID        NO: 93.

In other aspects, the effector-deficient AT-10 antibody comprises avariable heavy chain sequence comprising a sequence that is at least75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequenceshown in SEQ ID NO: 8, and a variable light chain sequence comprising asequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the sequence shown in SEQ ID NO: 10.

In other aspects, the effector-deficient AT-10 antibody comprises:

-   -   a. a heavy chain sequence that is at least 75%, 80%, 85%, 90%,        95%, 96%, 97%, 98%, or 99% identical to the sequence shown in        SEQ ID NO: 16 or SEQ ID NO: 18; and    -   b. a light chain sequence that is at least 75%, 80%, 85%, 90%,        95%, 96%, 97%, 98%, or 99% identical to the sequence shown in        SEQ ID NO: 22.

In another embodiment, the effector-deficient humanized AT-10 antibodycomprises a variable heavy chain sequence comprising a sequence that isat least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to thesequence shown in SEQ ID NO: 12; and a variable light chain sequencecomprising a sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 14.

In another aspect, the effector-deficient humanized AT-10 antibodycomprises:

a heavy chain sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 20; and

a light chain sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 24.

2. Effector-Deficient IV.3 Monoclonal Antibodies

In one embodiment, the effector-deficient anti-CD32a antibody is aneffector-deficient IV.3 antibody. In one aspect, the IV.3 antibodycomprises:

-   -   a. a heavy chain variable region CDR1 sequence comprising a        sequence that is at identical to the sequence NYGMN (SEQ ID        NO: 25) or GYTFTNYG (SEQ ID NO: 79), or is a sequence having 1        or 2 amino acid differences as compared to the sequence NYGMN        (SEQ ID NO: 25) or GYTFTNYG (SEQ ID NO: 79);    -   b. a heavy chain variable region CDR2 sequence comprising a        sequence that is identical to the sequence WLNTYTGESIYPDDFKG        (SEQ ID NO: 26) or LNTYTGES (SEQ ID NO: 80), or is a sequence        having 1 or 2 amino acid differences as compared to the sequence        WLNTYTGESIYPDDFKG (SEQ ID NO: 26) or LNTYTGES (SEQ ID NO: 80);    -   c. a heavy chain variable region CDR3 sequence comprising a        sequence that is identical to the sequence GDYGYDDPLDY (SEQ ID        NO: 27) or ARGDYGYDDPLDY (SEQ ID NO: 81), or is a sequence        having 1 or 2 amino acid differences as compared to the sequence        GDYGYDDPLDY (SEQ ID NO: 27) or ARGDYGYDDPLDY (SEQ ID NO: 81);    -   d. a light chain variable region CDR1 sequence comprising a        sequence that is identical to the sequence RSSKSLLHTNGNTYLH (SEQ        ID NO: 28) or KSLLHTNGNTY (SEQ ID NO: 82 and SEQ ID NO: 100), or        is a sequence having 1 or 2 amino acid differences as compared        to the sequence RSSKSLLHTNGNTYLH (SEQ ID NO: 28) or KSLLHTNGNTY        (SEQ ID NO: 82 and SEQ ID NO: 100);    -   e. a light chain variable region CDR2 sequence comprising a        sequence that is identical to the sequence RMSVLAS (SEQ ID        NO: 29) or RMS (SEQ ID NO: 83 and SEQ ID NO: 101), or is a        sequence having 1 or 2 amino acid differences as compared to the        sequence RMSVLAS (SEQ ID NO: 29) or RMS (SEQ ID NO: 83 and SEQ        ID NO: 101); and    -   f. a light chain variable region CDR3 sequence comprising a        sequence that is identical to the sequence MQHLEYPLT (SEQ ID        NO: 30) or MQHLEYPLT (SEQ ID NO: 84 and SEQ ID NO: 102), or is a        sequence having 1 or 2 amino acid differences as compared to the        sequence MQHLEYPLT (SEQ ID NO: 30) or MQHLEYPLT (SEQ ID NO: 84        and SEQ ID NO: 102).

In other aspects, the effector-deficient IV.3 antibody comprises avariable heavy chain sequence comprising a sequence that is at least75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequenceshown in SEQ ID NO: 32; and a variable light chain sequence comprising asequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the sequence shown in SEQ ID NO: 34.

In other aspects, the effector-deficient IV.3 antibody comprises:

-   -   a. a heavy chain sequence comprising a sequence that is at least        75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the        sequence shown in SEQ ID NO: 43 or SEQ ID NO: 45, and    -   b. a light chain sequence comprising a sequence that is at least        75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the        sequence shown in SEQ ID NO: 51.

In one embodiment, the effector-deficient IV.3 antibody is a humanizedantibody comprising a variable heavy chain sequence comprising asequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the sequence shown in SEQ ID NO:36 or SEQ ID NO: 38; and avariable light chain sequence comprising a sequence that is at least75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequenceshown in SEQ ID NO: 41 or SEQ ID NO: 85.

In certain embodiments, the effector-deficient humanized IV.3 antibodycomprises:

-   -   a. a heavy chain sequence that is at least 75%, 80%, 85%, 90%,        95%, 96%, 97%, 98%, or 99% identical to the sequence shown in        SEQ ID NO: 47 or SEQ ID NO: 49; and    -   b. a light chain sequence that is at least 75%, 80%, 85%, 90%,        95%, 96%, 97%, 98%, or 99% identical to the sequence shown in        SEQ ID NO: 53 or SEQ ID NO: 87.

3. Effector-Deficient MDE-8 Monoclonal Antibodies

In some aspects, the effector-deficient anti-CD32a antibody is aneffector-deficient MDE-8 antibody. In some aspects theeffector-deficient MDE-8 antibody comprises:

-   -   a. a heavy chain variable region CDR1 sequence comprising a        sequence that is identical to the sequence SYGMH (SEQ ID NO: 54)        or GFTFSSY (residues 1-7 of SEQ ID NO: 94), or is a sequence        having 1 or 2 amino acid differences as compared to the sequence        SYGMH (SEQ ID NO: 54) or GFTFSSY (residues 1-7 of SEQ ID NO:        94);    -   b. a heavy chain variable region CDR2 sequence comprising a        sequence that is identical to the sequence VIWYDGSNYYYTDSVKG        (SEQ ID NO: 55) or IWYDGSNY (SEQ ID NO: 95), or is a sequence        having 1 or 2 amino acid differences as compared to the sequence        VIWYDGSNYYYTDSVKG (SEQ ID NO: 55) or IWYDGSNY (SEQ ID NO: 95;    -   c. a heavy chain variable region CDR3 sequence comprising a        sequence that is identical to the sequence DLGAAASDY (SEQ ID        NO: 56) or ARDLGAAASDY (SEQ ID NO: 96), or is a sequence having        1 or 2 amino acid differences as compared to the sequence        DLGAAASDY (SEQ ID NO: 56) or ARDLGAAASDY (SEQ ID NO: 96);    -   d. a light chain variable region CDR1 sequence comprising a        sequence that is identical to the sequence RASQGINSALA (SEQ ID        NO: 57) or QGINSA (SEQ ID NO: 97), or is a sequence having 1 or        2 amino acid differences as compared to the sequence RASQGINSALA        (SEQ ID NO: 57) or QGINSA (SEQ ID NO: 97);    -   e. a light chain variable region CDR2 sequence comprising a        sequence that is identical to the sequence DASSLES (SEQ ID        NO: 58) or DAS (SEQ ID NO: 98), or is a sequence having 1 amino        acid differences as compared to the sequence DASSLES (SEQ ID        NO: 58) or DAS (SEQ ID NO: 98); and    -   f. a light chain variable region CDR3 sequence comprising a        sequence that is identical to the sequence QQFNSYPHT (SEQ ID        NO: 59) or QQFNSYPHT (SEQ ID NO: 99), or is a sequence having 1        or 2 amino acid differences as compared to the sequence        QQFNSYPHT (SEQ ID NO: 59) or QQFNSYPHT (SEQ ID NO: 99).

In other aspects, the effector-deficient MDE-8 antibody comprises avariable heavy chain sequence comprising a sequence that is at least75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequenceshown in SEQ ID NO: 61; and a variable light chain sequence comprising asequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the sequence shown in SEQ ID NO: 63.

In one embodiment, the effector-deficient anti-CD32a antibody is aneffector-deficient anti-MDE8 antibody comprising:

-   -   a. a heavy chain sequence that is at least 75%, 80%, 85%, 90%,        95%, 96%, 97%, 98%, or 99% identical to the sequence shown in        SEQ ID NO: 65 or SEQ ID NO: 67; and    -   b. a light chain sequence that is at least 75%, 80%, 85%, 90%,        95%, 96%, 97%, 98%, or 99% identical to the sequence shown in        SEQ ID NO: 69.

The antibodies of the composition and method embodiments may be fullyhuman, humanized, chimeric, recombinant, or synthetic.

In some aspects, the invention comprises an isolated antibody thatcompetes for binding to CD32a with an effector-deficient antibodydisclosed herein.

In some aspects, the invention comprises a pharmaceutical compositioncomprising an effector-deficient anti-CD32a antibody as describedherein.

In one embodiment, the effector-deficient anti-CD32a antibody is aneffector-deficient MDE-8, IV.3, or AT-10 monoclonal antibody. In oneembodiment, the effector-deficient MDE-8, IV.3, or AT-10 monoclonalantibody is humanized.

An effector deficient anti-CD32a monoclonal antibody that specificallybinds CD32a comprising at least a portion of an Fc domain that ismutated at one or more amino acids, wherein the mutation preventsFc-mediated binding to CD16, CD32, or CD64 IgG receptors is encompassed.

b. Further Antibody Embodiments

The antibodies in the composition and method embodiments may exhibit anyor all of the following functional features:

-   -   a. the antigen-binding portions of the antibodies bind human        CD32a with an equilibrium affinity constant value (“K_(D)”)        stronger (less than) than 10⁻⁸ M when in aqueous solution;    -   b. the antigen-binding portions of the antibodies bind human        CD32 where such binding inhibits stable interactions between        such bound-CD32 and the Fc-region of any human or therapeutic        IgG molecule where such human or therapeutic IgG molecule is        either: (1) bound in a Fab-dependent manner to at least one        antigen molecule, or (2) clustered into an assembly of at least        two such human or therapeutic IgG molecules, or (3) localized to        a surface in such a manner so as to restrict aqueous diffusion        of the human or therapeutic IgG molecule;    -   c. the antibodies include at least a portion of an Fc-region,        and either lack the capacity, or have reduced capacity, for        Fc-region binding to human IgG receptors (FcgammaRs) of        classical types I (CD64), II (CD32), or III (CD16), where such        reduced or absent binding is comparatively more than 20%, 30%,        40%, 45%, or 50% weaker than that of corresponding naturally        occurring classical IgG-Fc-regions (i.e., either of IgG1, IgG2,        IgG3, or IgG4), where any such classical IgG-Fc-region exhibits        binding to CD16, CD32, or CD64.

The antibodies in the composition and method embodiments may exhibit anyor all of the following structural features:

-   -   a. The antibodies comprise, consist, or consist essentially of        (in terms of amino acid composition) the following arrangement        of a total of four polypeptides per single IgG molecule:        -   i. two heavy chain polypeptides covalently bound together by            at least two cysteine-to-cysteine disulfide bonds, wherein            such interchain disulfide bonds are located in or near the            hinge region, and wherein such heavy chain polypetpides are            of the IgG isotype (class 1, 2, 3, or 4, or any hybrid            version comprising segments of the same) of heavy chain            immunoglobulin molecule, and where each such heavy chain            polypeptide is comprised of at least a portion of one            variable domain (VH) and one, two, or three constant domains            (CH1, CH2, and CH3) or portions thereof. An example of a            hybrid constant region IgG heavy chain molecule would be one            having an IgG1 CH1 domain with a hinge region derived from            IgG1 and the remaining carboxy-terminal portion of the            polypeptide derived from that of the CH2 and CH3 domains of            the IgG2 heavy chain;        -   ii. two light chain polypeptides covalently bound each            (individually) to a single heavy chain polypeptide (of item            [i] immediately above), wherein such covalent bond consists            of at least one cysteine-to-cysteine interchain disulfide            bond between a single said light chain and a single said            heavy chain polypeptide, and wherein such light chain            polypeptides are of the kappa or the lambda type of light            chain immunoglobulin molecules, each of which comprising,            consisting, or consisting essentially of at least one            variable domain (VL) and at least one light chain constant            domain;    -   b. The antibodies may have an apparent molecular mass (as        determined by SDS-PAGE analysis using 8%-12% polyacrylamide gels        under non-reducing conditions) greater than about 100,000        daltons and less than about 250,000 daltons, greater than about        120,000 and less than about 180,000 daltons, or about 140,000 to        165,000 daltons, in apparent molecular mass;    -   c. The antibodies may have heavy chain constant regions with        amino acid compositions that are at least 85%, 90%, 95%, 96%,        97%, 98%, or 99% identical to the heavy chain constant regions        of naturally occurring human IgG isotype molecules of class 1        (IgG1), 2 (IgG2), 3 (IgG3), or 4 (IgG4), wherein such identity        is determined for each amino acid of the antibodies compared to        each corresponding position naturally occurring in human IgG        heavy chains of any isotype, as found by genetic sequencing or        as reported in the relevant literature or as found in any        therapeutic IgG antibody used to treat human patients, wherein        such identity comparison allows sufficient sequence gap-lengths        and a sufficient quantity of gaps so as to maximize identity        between compared polypeptides. The composition of said naturally        occurring human antibody molecules includes any and all        allotypic variants (see, e.g., Jefferis R, Lefranc M P. Human        immunoglobulin allotypes: possible implications for        immunogenicity (2009 July-August) MAbs 1:332; PubMed ID:        20073133);    -   d. The antibodies may have light chain constant regions with        amino acid compositions that at least 85%, 90%, 95%, 96%, 97%,        98%, or 99% identical to the light chain constant regions of        naturally occurring human kappa or lambda type molecules,        wherein such identity is determined for each amino acid of the        antibodies compared to each corresponding position naturally        occurring in human light chains as found by genetic sequencing        or as reported in the relevant literature or as found in any        therapeutic antibody used to treat human patients, wherein such        identity comparison allows sufficient sequence gap-lengths and a        sufficient quantity of gaps so as to maximize identity between        compared polypeptides. The composition of said naturally        occurring human antibody molecules includes any and all possible        allotypic variants (see, e.g., Jefferis R, Lefranc M P. Human        immunoglobulin allotypes: possible implications for        immunogenicity (2009 July-August) MAbs 1:332; PubMed ID:        20073133);    -   e. The antibodies may comprise, consist, or consist essentially        of heavy chain variable (VH) and light chain variable (VL)        domains derived from a mammalian source (e.g., human, primate,        rabbit, ruminant, mouse or other rodent). In cases where the VH        or the VL coding source is not human, the antibody may be either        chimeric (due to the presence of human constant regions) or        humanized (due to the grafting of non-human amino acid sequences        onto a human framework variable region);    -   f. In one embodiment, the antibodies are not conjugated to any        of the following: (1) a cytotoxin (e.g., vincristine), (2) a        radioactive substance (e.g., ¹¹¹indium), (3) an imaging agent        (e.g., fluorescein), (4) a small molecule therapeutic drug        (e.g., bleomycin), (5) a therapeutic non-antibody polypeptide        (e.g., interferon-gamma), (6) an enzyme (e.g., a        peroxidase), (7) a vaccine-substance (e.g., a viral        polypeptide), or (8) a polyethylene glycol molecule (e.g., PEG).    -   g. In one embodiment, the antibodies are conjugated to any of        the following: (1) a cytotoxin (e.g., vincristine), (2) a        radioactive substance (e.g., ¹¹¹indium), (3) an imaging agent        (e.g., fluorescein), (4) a small molecule therapeutic drug        (e.g., bleomycin), (5) a therapeutic non-antibody polypeptide        (e.g., interferon-gamma), (6) an enzyme (e.g., a        peroxidase), (7) a vaccine-substance (e.g., a viral        polypeptide), or (8) a polyethylene glycol molecule (e.g., PEG).

The antibodies in the composition and method embodiments may exhibit anyor all of the following structure-function correlates:

-   -   a. The antibodies may comprise, consist, or consist essentially        of two CD32 binding domains that derive from the variable (Fab)        regions formed by each of the two heavy-light chain pairs, and        because of this divalent structure have the capacity to bind        either one or two antigen epitopes;    -   b. The Fc region of the antibodies is either reduced in its        ability, or completely lacking the ability, to bind human CD16,        CD32, or CD64 IgG receptors, wherein such reduced IgG receptor        binding activity is the result of either (1) fusion (e.g.,        hybridization) of two or more IgG-Fc-region polypeptide        sequences, (2) enzymatic modification of Fc-region carbohydrate        molecules (e.g., modification or removal of a carbohydrate        molecule from the asparagine residue located at position 297 in        the EU index of Kabat), or (3) engineered amino acid mutations        at one or more positions in the constant region of the IgG heavy        chain, wherein such engineered mutations reduce or eliminate        Fc-dependent binding to the following types of classical human        IgG receptors: CD16, CD32, and CD64;    -   c. The antibodies, when bound to human CD32, form stable immune        complexes that inhibit the capacity of the Fc region of other        IgG antibodies to cause said CD32 molecules to directly induce        inflammatory cellular reactions, wherein such other IgG        antibodies are either: (1) bound in a Fab-dependent manner to at        least one antigen molecule, or (2) clustered into an assembly of        at least two such IgG molecules, or (3) localized to a surface        in such a manner so as to restrict aqueous diffusion of said IgG        molecule.

Nucleic Acids, Vectors, and Host Cells

The invention also provides a synthetic or recombinant nucleic acidsequence encoding any of the antibodies described herein. Such nucleicacid is, for instance, isolated from a B-cell that is capable ofproducing an antibody described herein. Such nucleic acids encode theheavy and light chain sequences set forth herein. Alternatively, suchnucleic acids encode heavy and light chain sequences comprising theheavy and light chain CDRs, respectively, set forth herein. In someembodiments, the nucleic acids will encode functional parts of theantibodies described herein. Due to the degeneracy of the nucleic acidcode, multiple nucleic acids will encode the same amino acid and all areencompassed herein. Certain encompassed nucleic acids are described inTables 1-5.

In some aspects, the invention comprises a vector comprising a nucleicacid molecule as described herein. In some embodiments, the inventioncomprises a host cell comprising a nucleic acid molecule as describedherein.

In some aspects, the invention comprises a nucleic acid moleculeencoding at least one antibody disclosed herein.

Methods of Making Antibodies

In one embodiment, a method of making an effector-deficient anti-CD32aantibody is provided. In one aspect the method comprises culturing ahost cell comprising a nucleic acid encoding an effector-deficientanti-CD32a antibody and isolating a secreted antibody. The nucleic acidencoding the effector-deficient anti-CD32a antibody may be any nucleicacid described in Tables 1-5 or fragments or variants thereof.

In one embodiment, a host cell expressing an effector-deficientanti-CD32a antibody is encompassed. The host cell may be a mammaliancell. Non-limiting examples include host cells derived from a humanindividual, rodent, rabbit, llama, pig, cow, goat, horse, ape, orgorilla. In one embodiment, said host cell comprises a human cell, amurine cell, a rabbit cell and/or a llama cell.

In one embodiment, a host cell may comprise Chinese hamster ovary (CHO)cell line, 293(T) cells, COS cells, NS0 cells and other cell lines knownin the art and comprise nucleic acid sequences encoding the antibodydescribed herein. Host cells may be adapted to commercial antibodyproduction (“producer cell”). Proliferation of said producer cellresults in a producer cell line capable of producing effector-deficientanti-CD32a antibodies. A producer cell line may be suitable forproducing compounds for use in humans. Hence, said producer cell linemay be free of pathogenic agents such as pathogenic micro-organisms.

Further provided is a method for producing antibodies which are capableof specifically binding CD32a, wherein the antibody prevents theactivation of CD32a by immobilized IgG or prevents activation of CD32 byIgG immune complexes, the method comprising: producing anantibody-producing cell capable of producing said effector-deficientantibodies and obtaining antibodies produced by said antibody producingcell.

An isolated or recombinant antibody, as well as an isolated orrecombinant host cell, obtainable by one of the methods provided herein,or a functional equivalent thereof, is also provided.

In one embodiment, the antibodies were produced by obtaining nucleicacid molecules coding for the variable region of light chain and heavychains of anti-CD32a antibodies (IV.3, AT-10, and MDE-8). For example,the antibodies may be obtained: 1) from hybridoma cell lines by ReverseTranscription-Polymerase Chain Reaction (RT-PCR) using ribonucleic acid(RNA) isolated from these cell lines and oligo primers directed to the5′ leader coding sequence and 5′ constant chain coding sequence, or 2)by producing synthetic molecules (by commercially available means)containing the known nucleic acid sequences of variable regions of lightchain and heavy chains of anti-CD32a antibodies.

In one embodiment, nucleic acid molecules coding for humanized variableregions of light chains and heavy chains of anti-CD32a antibodies wereobtained by producing synthetic molecules (by commercially availablemeans) containing the known nucleic acid sequences with the modificationdescribed herein.

In one embodiment, nucleic acid molecules coding for the variable regionof light chains and heavy chains of anti-CD32a antibodies (IV.3, AT-10,and MDE-8) were cloned into commercially available plasmid vectors,pFUSE, that contain the respective nucleic acid sequences coding for thelight chain constant region (immunoglobulin kappa), and the heavy chainconstant regions (human IgG1 or human IgG2).

In one embodiment, effector-deficient anti-CD32a antibodies wereproduced by creating nucleic acid mutations by site-directed mutagenesison the heavy chain constant regions coding sequences of pFUSE plasmids.

In one embodiment, anti-CD32a antibodies were produced by transfectinghuman embryonic kidney cells (e.g. Expi293 cells) with pFUSE plasmidvectors containing nucleic acid molecules coding for variable regions aswell as constant regions of light and heavy antibody chains. In someaspects, the nucleic acid molecules coded for chimeric, humanized, orhuman anti-CD32a mAbs, in IgG1 or IgG2 isotype subclass, in native(effector-competent) or mutated (effector-deficient) format.

In one embodiment, anti-CD32a antibodies secreted by transfected cellswere purified from culture media by Protein G column purification anddialized in buffered saline prior to use.

Pharmaceutical Compositions

The invention comprises a pharmaceutical composition comprising at leastone effector-deficient anti-CD32a antibody as described herein and apharmaceutically acceptable excipient. In some embodiments, thepharmaceutical composition further comprises an additional active agent.

In certain embodiments, the pharmaceutical composition will bedetermined by one skilled in the art depending upon, for example, theintended route of administration, delivery format and desired dosage.See, for example, Remington's Pharmaceutical Sciences, 18th Edition, A.R. Gennaro, ed., Mack Publishing Company (1995). In certain embodiments,such compositions may influence the physical state, stability, rate ofin vivo release and rate of in vivo clearance of the antibodies of theinvention.

In certain embodiments, the excipient in the pharmaceutical compositioncan be either aqueous or non-aqueous in nature. For example, in certainembodiments, a suitable excipient can be water for injection,physiological saline solution or artificial cerebrospinal fluid,possibly supplemented with other materials common in compositions forparenteral administration. In some embodiments, the saline comprisesisotonic phosphate-buffered saline. In certain embodiments, neutralbuffered saline or saline mixed with serum albumin are further exemplaryexcipients. In certain embodiments, pharmaceutical compositions compriseTris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5,which can further include sorbitol or a suitable substitute therefore.In certain embodiments, a composition comprising an effector-deficientantibody as described herein, with or without at least one additionaltherapeutic agents, can be prepared for storage by mixing the selectedcomposition having the desired degree of purity with optionalformulation agents (Remington's Pharmaceutical Sciences, supra) in theform of a lyophilized cake or an aqueous solution. Further, in certainembodiments, a composition comprising an effector-deficient antibody asdescribed herein, with or without at least one additional therapeuticagents, can be formulated as a lyophilizate using appropriate excipientssuch as sucrose.

The antibodies/compositions of the present invention may be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques.

CD32a-Mediated Mediated Diseases and Disorders

CD32a-mediated diseases and disorders include heparin-inducedthrombocytopenia (HIT), immune thrombocytopenic purpura (ITP),antiphospholipid syndrome (APS), thrombosis associated with autoimmunityor with certain drugs (e.g., heparin) and antibody therapies (e.g.,anti-VEGF or anti-CD40L immunotherapies), transfusion or organtransplantation reactions, certain viral infections, rheumatoidarthritis (RA), psoriasis, psoriatic arthritis, inflammatory boweldisease, osteoarthritis, systemic lupus erythematous (SLE), asthma,allergic rhinitis, lupus nephritis, antibody-mediated anemias,anaphylaxis and airway inflammation. See, e.g., Gillis C et al.Contribution of Human FcgammaRs to Disease with Evidence from HumanPolymorphisms and Transgenic Animal Studies (2014 May 30) Front Immunol5:254; PubMed ID: 24910634; Bruhns P. Properties of mouse and human IgGreceptors and their contribution to disease models (2012 Jun. 14) Blood,119(24):5640-9, PubMed ID: 22535666; and Hogarth P M and Pietersz G A,Fc receptor-targeted therapies for the treatment of inflammation, cancerand beyond (2012 Mar. 30) Nat Rev Drug Discov 11(4):311-31, PubMed ID:22460124.

In one embodiment, the CD32a-mediated disease or disorder isthrombocytopenia. Thrombocytopenia is characterized by a drop incirculating platelets. In one embodiment, thrombocytopenia is defined asa platelet count less than the lower limit of normal (usually taken as150×10⁹/L). In other embodiments, thrombocytopenia is defined as a fallin the number of circulating platelets. For example, a fall in theplatelet count of 30-50% or more, following administration of heparin,may be a symptom of heparin-induced thrombocytopenia, even if theplatelet count does not fall below 150×10⁹/L. (Warkentin T E. Clinicalpresentation of heparin-induced thrombocytopenia (October 1998) SeminHematol 35(4 Suppl 5):9-16; discussion 35-6; PubMed ID: 9855179). Theplatelet count is typically measured by electronic counting methods, andusually as part of a Complete Blood Count (CBC). Methods for treatingthrombocytopenia with any one of or a combination of theeffector-deficient antibodies described herein, alone or in combinationwith other therapies, are encompassed.

In another embodiment, the CD32a-mediated disease or disorder isIgG-mediated thrombosis. In one embodiment, IgG-mediated thrombosis isthrombosis caused by IgG immune complexes or by immobilzed IgG (see,e.g., Reilly M P et al. Heparin-induced thrombocytopenia/thrombosis in atransgenic mouse model requires human platelet factor 4 and plateletactivation through FcgammaRIIA. Blood. 2001 Oct. 15; 98(8):2442-7.PubMed ID: 11588041; and also Taylor S M et al. Thrombosis and shockinduced by activating antiplatelet antibodies in human FcgammaRIIAtransgenic mice: the interplay among antibody, spleen, and Fc receptor.Blood. 2000 Dec. 15; 96(13):4254-60. PubMed ID: 11110699, respectively).Methods for treating IgG-mediated thrombosis with any one of or acombination of the effector-deficient antibodies described herein areencompassed. A method for treating IgG-mediated thrombosis comprisingadministering one or a combination of an effector-deficient antibody asdescribed herein, alone or in combination with other therapies, whereinIgG-thrombosis is any thrombosis caused by IgG immune complexes or byimmboilized IgG, is encompassed.

In some aspects, the CD32a-mediated disease or disorder is caused, atleast in part, by activation of CD32a on or in cells (Hogarth P M et al.Fc receptor-targeted therapies for the treatment of inflammation, cancerand beyond (30 Mar. 2012) Nat Rev Drug Discov 11:311; PubMed ID:22460124), including platelets, monocytes, neutrophils, basophils,eosinophils, macrophages, dendritic cells (Boruchov A M et al.Activating and inhibitory IgG Fc receptors on human DCs mediate opposingfunctions (October 2005) J Clin Invest 115:2914; PubMed ID: 16167082),mast cells, and dermal microvascular endothelial cells (Gröger M et al.Dermal microvascular endothelial cells express CD32 receptors in vivoand in vitro (15 Feb. 1996) J Immunol 156:1549; PubMed ID: 8568259). Insome other aspects, the CD32a-mediated disease or disorder is caused, atleast in part, by activation of CD32a on malignant cells, e.g.,Hodgkin's disease, non-Hodgkin's lymphoma, Burkitt's lymphoma,anaplastic large cell lymphoma, cutaneous T-cell lymphomas, nodularsmall cleaved-cell lymphomas, lymphocytic lymphomas, peripheral T-celllymphomas, Lennert's lymphomas, immunoblastic lymphomas, T-cellleukemias/lymphomas, adult T-cell leukemia, follicular lymphomas,diffuse large cell lymphomas of B lineage, angioimmunoblasticlymphadenopathy (AILD)-like T-cell lymphoma, HIV-associated body cavitybased lymphomas, Embryonal carcinomas, undifferentiated carcinomas ofthe rhino-pharynx, Castleman's disease, Kaposi sarcoma and other B-celllymphomas. Methods for treating a disease or disorder characterized byactivation of CD32a with any one of or a combination of theeffector-deficient antibodies described herein, alone or in combinationwith other therapies, are encompassed.

In one embodiment, the CD32a-mediated disease or disorder is an immune,autoimmune, allergic, or inflammatory disease or disorder. The immune,autoimmune, allergic, or inflammatory disorder may be rheumatoidarthritis (RA), psoriasis, psoriatic arthritis, inflammatory boweldisease, including Crohn's disease and ulcerative colitis,antiphospholipid syndrome (APS), atopic dermatitis, chronic inflammatorypulmonary disease, osteoarthritis, systemic lupus erythematous (SLE),lupus nephritis, systemic scelrosis, Graves' disease, Hashimoto'sthyroiditis, Wegner's granulomatosis, Omen's syndrome, chronic renalfailure, idiopathic thrombocytopenic purpura, insulin-dependent diabetesmellitus, acute infectious mononucleosis, HIV, herpes virus-associateddiseases, multiple sclerosis, hemolytic anemia, thyroiditis, stiff mansyndrome, pemphigus vulgaris, and myasthenia gravis, antibody-mediatedarthritis, or antibody-induced anemias or cytopenias. Methods fortreating an immune, autoimmune, allergic, or inflammatory disease ordisorder with any one of or a combination of the effector-deficientantibodies described herein are encompassed. Methods for treatingrheumatoid arthritis (RA), psoriasis, psoriatic arthritis, inflammatorybowel disease, including Crohn's disease and ulcerative colitis,antiphospholipid syndrome (APS), atopic dermatitis, chronic inflammatorypulmonary disease, osteoarthritis, systemic lupus erythematous (SLE),lupus nephritis, antibody-mediated arthritis, or antibody-inducedanemias or cytopenias with any one of or a combination of theeffector-deficient antibodies described herein, alone or in combinationwith other therapies, are encompassed.

The CD32a-mediated disease or disorder may be an immune complex-mediateddisease or disorder. Immune complex-mediated diseases or disorders arecharacterized by localized or systemic inflammatory processes thatdamage cells and tissues, as in the cases, for example, of inflammationcaused by IgG-induced release of Tumor Necrosis Factor alpha (TNF-alpha,an inflammatory cytokine) from monocytes in RA (Mathsson L et al. Immunecomplexes from rheumatoid arthritis synovial fluid induce FcgammaRIIadependent and rheumatoid factor correlated production of tumour necrosisfactor-alpha by peripheral blood mononuclear cells (2006) Arthritis ResTher 8:R64; PubMed ID: 16569263), or of kidney damage caused bypolymorphonuclear cells (neutrophils, basophils, eosinophils) in SLE andglomerulonephritis (Suzuki Y et al. Pre-existing glomerular immunecomplexes induce polymorphonuclear cell recruitment through an Fcreceptor-dependent respiratory burst: potential role in the perpetuationof immune nephritis (2003 Mar. 15) J Immunol 170:3243; PubMed ID:12626583; Rovin B H. The chemokine network in systemic lupuserythematous nephritis (2008 Jan. 1) Front Biosci 13:904; PubMed ID:17981599). Immune complex-mediated diseases or disorders includenumerous other acute and chronic conditions (Gillis C et al.Contribution of Human FcgammaRs to Disease with Evidence from HumanPolymorphisms and Transgenic Animal Studies (2014 May 30) Front Immunol5:254; PubMed ID: 24910634). Methods for treating an immunecomplex-mediated disease or disorder with any one of or any combinationof the effector-deficient antibodies described herein, alone or incombination with other therapies, are encompassed.

Diseases or disorders known to be associated with immune complexformation include rheumatoid arthritis (RA), systemic lupuserythematosus (SLE), heparin-induced thrombocytopenia (HIT), lupusnephritis, and APS. Methods for treating rheumatoid arthritis (RA),systemic lupus erythematosus (SLE), heparin-induced thrombocytopenia(HIT), lupus nephritis, and APS with any one of or any combination ofthe effector-deficient antibodies described herein, alone or incombination with other therapies, are encompassed.

The types of immune complexes associated with such diseases or disordersinclude circulating IgG immune complexes, deposited IgG immunecomplexes, and immobilized IgG immune complexes. Methods for treatingany disease or disorder characterized by circulating IgG immunecomplexes, deposited IgG immune complexes, or immobilized IgG immunecomplexes with any one of or any combination of the effector-deficientantibodies described herein, alone or in combination with othertherapies, are encompassed.

In one embodiment, a disease or disorder characterized by circulatingIgG immune complexes, deposited IgG immune complexes, or immobilized IgGimmune complexes includes RA and SLE characterized by circulating IgGimmune complexes. See, e.g., Zhao X et al. Circulating immune complexescontain citrullinated fibrinogen in rheumatoid arthritis (2008)Arthritis Res Ther 10:R94; PubMed ID: 18710572; Ohyama K et al. Immunecomplexome analysis of serum and its application in screening for immunecomplex antigens in rheumatoid arthritis (2011 June) Clin Chem 57:905;PubMed ID: 21482748; Soares N M et al. An improved anti-C3/IgG ELISA forquantification of soluble immune complexes (1 Mar. 2001) J ImmunolMethods 249:199; PubMed ID: 11226477; and Huber C et al. C3-containingserum immune complexes in patients with systemic lupus erythematosus:correlation to disease activity and comparison with other rheumaticdiseases (1989) Rheumatol Int 9:59; PubMed ID: 2814209). Methods fortreating RA and SLE, wherein the RA or SLE is characterized bycirculating IgG immune complexes with any one of or any combination ofthe effector-deficient antibodies described herein, alone or incombination with other therapies, are encompassed.

In one embodiment, a disease or disorder characterized by circulatingIgG immune complexes, deposited IgG immune complexes, or immobilized IgGimmune complexes includes RA, SLE, and APS characterized by IgG immunecomplexes deposited on circulating cells or particles or in tissues.See, e.g., Zhao X et al. Circulating immune complexes containcitrullinated fibrinogen in rheumatoid arthritis (2008) Arthritis ResTher 10:R94; PubMed ID: 18710572; Nielsen C T et al. Increased IgG oncell-derived plasma microparticles in systemic lupus erythematosus isassociated with autoantibodies and complement activation (April 2012)Arthritis Rheum 64:1227; PubMed ID: 22238051; and de Groot P G et al.The significance of autoantibodies against beta-2 glycoprotein I (Jul.12, 2012) Blood 120:266; PubMed ID: 22553312). Methods for treating RA,SLE, and APS, wherein the RA, SLE, or APS is characterized by IgG immunecomplexes deposited on circulating cells or particles or in tissues withany one of or any combination of the effector-deficient antibodiesdescribed herein, alone or in combination with other therapies, areencompassed.

In one embodiment, a disease or disorder characterized by circulatingIgG immune complexes, deposited IgG immune complexes, or immobilized IgGimmune complexes includes RA, SLE, HIT, and APS, wherein the RA, SLE,HIT, or APS is characterized by soluble or immobilized immune complexes.See, e.g., Ohyama K et al. Immune complexome analysis of serum and itsapplication in screening for immune complex antigens in rheumatoidarthritis (2011 June) Clin Chem 57:905; PubMed ID: 21482748; Ronnelid Jet al. Immune complexes from SLE sera induce IL10 production from normalperipheral blood mononuclear cells by an FcgammaRII dependent mechanism:implications for a possible vicious cycle maintaining B cellhyperactivity in SLE (January 2003) Ann Rheum Dis 62:37; PubMed ID:12480667; Cines D B et al. Heparin-induced thrombocytopenia: anautoimmune disorder regulated through dynamic autoantigenassembly/disassembly (February 2007) J Clin Apher 22:31; PubMed ID:17285619; and de Groot P G et al. The significance of autoantibodiesagainst beta-2 glycoprotein I (Jul. 12, 2012) Blood 120:266; PubMed ID:22553312. Methods for treating RA, SLE, HIT, or APS, wherein the RA,SLE, HIT, or APS is characterized by soluble or immobilized immunecomplexes with any one of or any combination of the effector-deficientantibodies described herein, alone or in combination with othertherapies, are encompassed.

Importantly, more than one type of the above-mentioned immune complexesmay be present simultaneously or at differing times in these and otherimmune complex diseases and disorders. Even in this scenario, theeffector-deficient antibodies described herein may be administered totreat one or all of the diseases and disorders.

In one embodiment, methods of treating diseases or disorderscharacterized by antibodies that bind PF4 complexes comprisingadministering any one of or any combination of the effector-deficientanti-CD32a monoclonal antibodies are encompassed. Antibodies to humanplatelet factor 4 (PF4) complexes have been identified in RA, APS, SLE,and HIT. See, e.g., Ohyama K et al. Immune complexome analysis of serumand its application in screening for immune complex antigens inrheumatoid arthritis (2011 June) Clin Chem 57:905; PubMed ID: 21482748;Sikara M P et al. Beta 2 Glycoprotein I binds platelet factor 4 (PF4):implications for the pathogenesis of antiphospholipid syndrome (Jan. 21,2010) Blood 115:713; PubMed ID: 19805618; Satoh T et al.Heparin-dependent and -independent anti-platelet factor 4 autoantibodiesin patients with systemic lupus erythematosus (2012 September)Rheumatology (Oxford) 51:1721 PubMed ID: 22718864; and Warkentin T E etal. HITlights: a career perspective on heparin-induced thrombocytopenia(2012 May) Am J Hematol 87:S92; PubMed ID: 22367928. Thus, in oneembodiment, methods of treating RA, APS, SLE, and HIT, wherein the RA,APS, SLE, or HIT is characterized by antibodies that bind PF4 complexes,comprising administering any one of or any combination of theeffector-deficient anti-CD32a monoclonal antibodies, either alone or incombination with existing therapies, are encompassed.

In HIT, anti-PF4 IgG antibodies are known to mediate thrombocytopeniaand thrombosis via platelet CD32a, where therapeutic amounts of heparin(where heparin is bound to PF4 antigen) play a key role in localizingHIT immune complexes to the platelet surface. See, e.g., Newman P M etal. Heparin-induced thrombocytopenia: new evidence for the dynamicbinding of purified anti-PF4-heparin antibodies to platelets and theresultant platelet activation (1 Jul. 2000) Blood 96:182; PubMed ID:10891449.

In one embodiment, the immune complex-mediated disease is ananti-therapeutic-antibody (ATA) response caused by administration of anon-anti-CD32a antibody or antigen-binding fragment thereof. Thenon-anti-CD32a antibody may be infliximab, adalimumab, the IgG-Fc-fusiontherapeutic, etanercept, certolizumab pegol, golimumab, etanercept,ustekinumab, bevacizumab, omalizumab, belimumab, or tabalumab. In thesemethod embodiments, the effector deficient anti-CD32a antibody may beadministered prior to, concurrently with, or following thenon-anti-CD32a monoclonal antibody.

In one embodiment, the immune complex-mediated disease or disorderoccurs in a patient being treated with a non-anti-CD32a monoclonalantibody for the treatment of RA, SLE, HIT, lupus nephritis, orantiphospholipid syndrome (APS). Methods for treating RA, SLE, HIT,lupus nephritis, or antiphospholipid syndrome (APS), wherein the patientis or has received a non-anti-CD32a monoclonal antibody, with any one ofor any combination of the effector-deficient antibodies describedherein, alone or in combination with other therapies, are encompassed.

In other embodiments, the disease or disorder is a hemostatic disorder.The hemostatic disorder may be selected from the group consisting ofantibody-mediated-thrombocytopenia, immune-mediated-thrombocytopenia(ITP), heparin-induced thrombocytopenia (HIT), and heparin-inducedthrombocytopenia with thrombosis (HITT). Methods for treating ahemostatic disorder comprising administering any one of or anycombination of the effector-deficient antibodies described herein isencompassed. Also encompassed are methods for treatingantibody-mediated-thrombocytopenia, immune-mediated-thrombocytopenia(ITP), heparin-induced thrombocytopenia (HIT), and heparin-inducedthrombocytopenia with thrombosis (HITT) comprising administering any oneof or any combination of the effector-deficient antibodies describedherein, alone or in combination with other therapies (e.g.,anticoagulants).

Also encompassed are methods for treating hemostatic disorders caused bytreatment of patients with IV-Ig comprising administering any one of orany combination of the effector-deficient antibodies described herein,where such effector-deficient antibodies are administered prior toIV-Ig, concurrently with IV-Ig, or subsequently to IV-Ig treatment.IV-Ig is useful for treating autoimmune and transplant patients, but isassociated with side effects such as thrombocytopenia and acute arterialand venous thrombosis, anaphylactic shock, transitory renal failure,increased risk of infection, and leucopenia. Thrombosis has beenincreasingly recognized in treatment with IV-Ig (Paran D et al. Venousand arterial thrombosis following administration of intravenousimmunoglobulins (July (2005) Blood Coagul Fibrinolysis 16:313; PubMedID: 15970713; Woodruff R K et al. Fatal thrombotic events duringtreatment of autoimmune thrombocytopenia with intravenous immunoglobulinin elderly patients (July 1986) Lancet 2:217; PubMed ID: 2873457).Serious thromboembolic events observed with IV-Ig use include deepvenous thrombosis (DVT), myocardial infarction (MI), pulmonary embolism(PE), central retinal vein occlusion, and cerebrovascular accidents(CVA). Pollreisz and colleagues showed that IVIg can induce activation,aggregation, degranulation, and inflammatory cytokine release fromplatelets in a CD32-dependent manner, and this IVIg-inducedCD32-dependent platelet activation was completely blocked by AT-10,demonstrating that platelet CD32 was both necessary and sufficient forIVIg-induced prothrombotic activity (Pollreisz A et al. Intravenousimmunoglobulins induce CD32-mediated platelet aggregation in vitro(September 2008) Br J Dermatol 159:578; PubMed PMID: 18565176).

In still other embodiments, the CD32a-mediated disease or disorder is anallergic disorder. The allergic disorder may be selected from the groupconsisting of ashtma, contact dermatitis, allergic rhinitis,anaphylaxis, and allergic reactions. Methods for treating allergicdisorder comprising administering any one of or any combination of theeffector-deficient antibodies described herein are encompassed.Likewise, methods for treating asthma, contact dermatitis, allergicrhinitis, anaphylaxis, and allergic reactions comprising administeringany one of or any combination of the effector-deficient antibodiesdescribed herein, alone or in combination with other therapies, areencompassed.

The presence of both the CD32 IgG receptor and the IgE receptor(Hasegawa S et al. Functional expression of the high affinity receptorfor IgE (FcepsilonRI) in human platelets and its' [sic] intracellularexpression in human megakaryocytes (April 1999) Blood 93:2543; PubMedID: 10194433) on the surface of human platelets indicates a vital linkbetween platelets and allergy, which is particularly evident inpulmonary inflammation, as occurs in asthma and chronic lung disease(Page C et al. Platelets and allergic inflammation (July 2014) Clin ExpAllergy 44:901; PubMed ID: 24708345). The link between CD32 and IgE hassimilarly been recognized for immature B-lymphocytes, where IV.3 orAT-10 blockade of CD32 on human tonsillar B-cells was shown to suppressboth inducible IgG and inducible IgE synthesis (Horejs-Hoeck J et al.Inhibition of immunoglobulin E synthesis through Fc gammaRII (CD32) by amechanism independent of B-cell receptor co-cross-linking (July 2005)Immunology 115:407; PubMed ID: 15946258). A mechanistic explanation forCD32/IgE synergy may have recently been identified in the capacity ofIV.3 and AT-10 to induce an anti-inflammatory state in CD32a-bearingcells (Ben Mkaddem S et al. Shifting Fc[gamma]RIIA-ITAM from activationto inhibitory configuration ameliorates arthritis (September 2014) JClin Invest 124:3945; PubMed PMID: 25061875). The role of CD32a inallergy may also be linked to disorders of hemostasis (Potaczek D P.Links between allergy and cardiovascular or hemostatic system (January2014) Int J Cardiol 170:278; PubMed ID: 24315352).

In one embodiment, effector-deficient anti-CD32a monoclonal antibodiesare used to suppress inflammation driven by reactions in cellsdisplaying CD32a, where such CD32a binds IgG molecules that areimmobilized on a surface, such as that of platelets or red blood cells.For example, immobilized IgG binds and activates platelet CD32a, leadingto adhesion and granule secretion, and this process has been shown to beblocked by IV.3 (Haimovich B et al. The FcgammaRII receptor triggerspp125FAK phosphorylation in platelets (July 1996) J Biol Chem 271:16332;PubMed ID: 8663117). Additionally, IgG-coated red blood cells arephagocytosed via CD32a, and this activity is inhibited by IV.3 (Wiener Eet al. Role of Fc gamma RIIa (CD32) in IgG anti-RhD-mediated red cellphagocytosis in vitro (September 1996) Transfus Med 6:235; PubMed ID:8885153). Additionally, IgG-coated cells are cleared in aCD32a-dependent manner in patients with SLE, where such mechanism isinhibited by IV.3 (Seres T et al. Correlation of Fc gamma receptorexpression of monocytes with clearance function by macrophages insystemic lupus erythematosus (September 1998) Scand J Immunol 48:307;PubMed ID: 9743218).

In one embodiment, effector-deficient anti-CD32a monoclonal antibodiesare used to suppress inflammation driven by reactions in cellsdisplaying CD32a, where such CD32a interacts with IgG molecules bound toself antigens, such as von Willebrand Factor (vWF), and localize to theCD32a-positive cell, leading to inflammatory activation that is known tobe inhibited by IV.3 (for example, see Hoylaerts M F et al. Recurrentarterial thrombosis linked to autoimmune antibodies enhancing vonWillebrand factor binding to platelets and inducing Fc gamma RIIreceptor-mediated platelet activation (April 1998) Blood 91:2810; PubMedID: 9531591). Thus, methods for suppressing inflammation comprisingadministering one or more effector-deficient anti-CD32a monoclonalantibodies, thereby suppressing inflammation, are encompassed.

In one embodiment, effector-deficient anti-CD32a monoclonal antibodiesare used to suppress inflammation driven by infectious viruses. Forexample, IV.3 is known to inhibit dengue virus infection of human mastcells (Brown M G et al. A dominant role for FcgammaRII inantibody-enhanced dengue virus infection of human mast cells andassociated CCL5 release (December 2006) J Leukoc Biol 80:1242; PubMedID: 16940332). Thus, methods for suppressing inflammation comprisingadministering one or more effector-deficient anti-CD32a monoclonalantibodies, wherein the inflammation is mediated by infectious viruses,thereby suppressing inflammation, are encompassed.

In one embodiment, effector-deficient anti-CD32a monoclonal antibodiesare used to suppress inflammation driven by infectious microbes. Forexample, staphylococcus aureus can cause infective endocarditis,inducing platelet-driven CD32a inflammatory reactivity, which isinhibited by IV.3 (Fitzgerald J R et al. Fibronectin-binding proteins ofStaphylococcus aureus, Streptococcus sanguinis, Streptococcus gordonii,Streptococcus oralis, and Streptococcus pneumoniae mediate activation ofhuman platelets via fibrinogen and fibronectin bridges to integrinGPIIb/IIIa and IgG binding to the FcgammaRIIa receptor (January 2006)Mol Microbiol 59:212; PubMed ID: 16359330; Arman M et al. Amplificationof bacteria-induced platelet activation is triggered by Fc[gamma]RIIA,integrin [alpha]IIb[beta]3, and platelet factor 4 (May 2014) Blood123:3166; PubMed ID: 24642751). Similarly, systemic inflammation,sepsis-associated vascular leakage, platelet activation, and coagulationdysfunction in gram-positive sepsis can be CD32a-mediated, and theseinflammatory processes are blocked by IV.3 (Sun D et al. Bacillusanthracis peptidoglycan activates human platelets through Fc[gamma]RIIand complement (July 2013) Blood 122:571; PubMed ID: 23733338). Thus,methods for suppressing inflammation comprising administering one ormore effector-deficient anti-CD32a monoclonal antibodies, wherein theinflammation is mediated by infectious microbes, thereby suppressinginflammation, are encompassed.

In one embodiment, effector-deficient anti-CD32a monoclonal antibodiesare administered as treatment to patients along with or as a replacementfor IV-Ig. Intravenous immunoglobulin (IgG), or “IV-Ig”, is approved bythe FDA for treatment of various autoimmune or inflammatory diseases,including Primary Humoral Immunodeficiency, Multifocal Motor Neuropathy,B-cell Chronic Lymphocytic Leukemia, Immune Thrombocytopenic Purpura,Kawasaki syndrome, Chronic Inflammatory Demyelinating Polyneuropathy.IVIg is also used to treat neonatal alloimmune thrombocytopenia,HIV-associated thrombocytopenia, autoimmune neutropenia, autoimmunehemolytic anemia, interstitial pneumonia or cytomegalovirus infection inbone marrow transplant patients, bullous pemphigoid, epidermolysisbullosa acquisita, mucous-membrane pemphigoid, necrotizing fasciitis,pemphigus foliaceus, pemphigus vulgaris, toxic epidermal necrolysis orStevens-Johnson syndrome, birdshot retinopathy, Guillain-Barré syndrome,Lambert-Eaton myasthenic syndrome, myasthenia gravis,opsoclonus-myoclonus, polyradiculoneuropathy, refractorydermatomyositis, refractory polymyositis, relapsing-remitting multiplesclerosis. Effector-deficient anti-CD32a monoclonal antibodies may beused to treat these conditions.

In each of the method embodiments, the CD32a-mediated disease ordisorder may be characterized by symptoms of shock. As used herein, theterm “shock” includes, but is not limited to, hypersensitivity reactionsof type I (i.e., mediated by IgE), type II (i.e., mediated byimmobilized IgG), or type III (i.e., mediated by IgG complexes),IgG-mediated thrombotic reactions, and IgG-mediated neurologicreactions. Methods for alleviating the symptoms of shock comprisingadministering any one of or any combination of the effector-deficientantibodies described herein, alone or in combination with othertherapies, are encompassed.

Exemplary Embodiments

In one embodiment, a method for treating a CD32a-mediated disease ordisorder in a human subject comprising administering a therapeuticallyeffective amount of an effector-deficient anti-CD32a monoclonal antibodyto a human subject, wherein the antibody comprises at least a portion ofan Fc region and is effector-deficient, thereby treating theCD32a-mediated disease or disorder is provided.

In one embodiment, the effector-deficient antibody satisfies both theIgG Immune Complex Test and the Immobilized IgG Test, and has an Fcregion that has been altered so as to reduce or eliminate Fc-binding toCD16, CD32, and/or CD64 type IgG receptors.

In any of the method embodiments described herein, the CD32a-mediateddisease or disorder may be an IgG-mediated hemostatic disorder. Thehemostatic disorder may be thrombosis with or without thrombocytopenia.The hemostatic disorder may be selected from the group consisting ofIgG-mediated-thrombocytopenia, immune-mediated-thrombocytopenia (ITP),antiphospholipid syndrome (APS), anti-platelet-antibody disorders,heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopeniawith thrombosis (HITT), cancer-induced platelet activation,cancer-induced hypercoagulability, platelet-mediated tumor cellmetastasis, and platelet-mediated cancer metastasis.

In any of the method embodiments described herein, the CD32a-mediateddisease or disorder may be characterized by IgG-Fc-mediated activationof CD32a on platelets, monocytes, neutrophils, basophils, eosinophils,macrophages, dendritic cells, synovial cells, mast cells, or dermalmicrovascular endothelial cells.

In any of the method embodiments described herein, the CD32a-mediateddisease or disorder may be an IgG-mediated immune, autoimmune, orinflammatory disease or disorder. The IgG-mediated immune, autoimmune orinflammatory disorder may be selected from the group consisting ofrheumatoid arthritis (RA), psoriasis, psoriatic arthritis, ankylosingspondylitis, inflammatory bowel disease, ulcerative colitis, Crohn'sdisease, antiphospholipid syndrome (APS), osteoarthritis, systemic lupuserythematous (SLE), lupus nephritis, IgG antibody-induced anemia, andIgG-mediated cytopenia.

In any of the method embodiments described herein, the CD32a-mediateddisease or disorder may be an IgG immune complex-mediated disease ordisorder. The IgG immune complex-mediated disease may be ananti-therapeutic-antibody (ATA) response caused by administration of anon-anti-CD32a monoclonal antibody or fragment thereof. In any of themethod embodiments described herein, the non-anti-CD32a antibody may beinfliximab, adalimumab, certolizumab pegol (antibody-like), golimumab,etanercept (antibody-like), ustekinumab, omalizumab, or bevacizumab. Inany of the method embodiments described herein, the effector deficientanti-CD32a antibody may be administered prior to, concurrently with, orfollowing the non-anti-CD32a monoclonal antibody. In any of the methodembodiments, alone or in combination with other methods, the IgG immunecomplex-mediated disease or disorder may occur in a patient beingtreated with a non-anti-CD32a monoclonal antibody for the treatment ofrheumatoid arthritis, systemic lupus erythematosus (SLE), lupusnephritis, or inflammatory bowel disease (IBD), including ulcerativecolitis and Crohn's disease.

In any of the method embodiments, the CD32a-mediated disease or disordermay be characterized by IgG localized on the surface of cellscirculating in the blood of the human subject. The circulating cell typemay be one or more of the following: platelets, erythrocytes, monocytes,neutrophils, basophils, eosinophils, B-lymphocytes, macrophages, mastcells, leukemia cells, or microbes such as viruses, bacteria, fungal, orparasitic organisms. In any of the method embodiments, the disease ordisorder that is characterized by IgG localized on the surface of cellsmay be one or more of the following: thrombocytopenia, leukopenia,neutropenia, lymphopenia, monocytopenia, anemia, hemolytic anemia, orsepsis.

In some embodiments, a method for treating antibody-mediated allergic orhypersensitivity reactions of type I, type II, or type III in a humansubject comprising: administering a therapeutically effective amount ofan effector-deficient anti-CD32a monoclonal antibody to a human subject,wherein the antibody comprises at least a portion of an Fc region and iseffector-deficient, thereby treating the antibody-mediated allergic orhypersensitivity reactions of type I, type II, or type III, is provided.In this and any of the method embodiments, or in any combination ofmethod embodiments, the effector-deficient antibody satisfies both theIgG Immune Complex Test and the Immobilized IgG Test, and has an Fcregion that has been altered so as to reduce or eliminate Fc-binding toCD16, CD32, and/or CD64 type IgG receptors. In any of the methodembodiments, the allergic disorder may be selected from the groupconsisting of atopy, contact dermatitis, allergic rhinitis, systemicanaphylaxis, localized anaphylaxis as exhibited in hay fever, asthma,hives, food allergies, eczema, allergic reactions to vaccines, allergicreactions to foods, allergic reactions to insect products, allergicreactions to drugs, allergic reactions to mold spores, allergicreactions to animal hair and dander, allergic reactions to latex, bloodtransfusion reactions, platelet transfusion reactions, erythrocytetransfusion reactions, erythroblastosis fetalis, hemolytic anemia, serumsickness, infusion reactions, necrotizing vasculitis,glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus,and allergic reactions to microorganisms.

In each of the method embodiments described herein, including anycombination of the various embodiments, the effector-deficientanti-CD32a antibody may be an effector-deficient MDE-8, IV.3, or AT-10monoclonal antibody, and the monoclonal antibody may be human orhumanized.

In each of the method embodiments described herein, including anycombinations of the various method embodiments, the MDE-8, IV.3, andAT-10 monoclonal antibodies may comprise the six CDRs for each antibody,as described herein and in the sequence listing, or may comprise asequence having 1 or 2 amino acid differences in the CDRs as recitedherein and in the sequence listing.

An effector deficient anti-CD32a monoclonal antibody that specificallybinds human CD32a, wherein the antibody comprises at least a portion ofan Fc region that is effector deficient, wherein the effector-deficientantibody comprises an altered Fc region that reduces or eliminatesFc-binding to CD16, CD32, and/or CD64 type IgG receptors, as compared toa non-altered control, is provided.

Definitions

As used herein, the term “Human CD32A mice,” “CD32A mice,” “transgenicCD32A mice,” and “transgenic human CD32A mice” are used interchangeably.CD32A mice have been previously described (McKenie et al., The role ofthe human Fc receptor FcgammaRIIA in the immune clearance of platelets:a transgenic mouse model. J Immunol. 1999 Apr. 1; 162(7):4311-8. PubMedID: 10201963).

Fc receptors (FcR) are leukocyte surface glycoproteins that specificallybind the Fc portion of antibodies. The receptors for IgG, that isFcgammaR, are the most widespread and diverse, the major types beingFcgammaRT (CD64), FcgammaRII (CD32) and FcgammaRIII (CD16). As usedherein, the term “CD32a” is synonymous with the activating type ofFcgammaRII and iterations thereof such as iterations using the Greekgamma symbol in lieu of “gamma”.

The term “antibody” refers to an intact immunoglobulin of any isotype,or a fragment thereof that can compete with the intact antibody forspecific binding to the target antigen, and includes, for instance,chimeric, humanized, fully human, and bispecific antibodies. An intactantibody may comprise at least two full-length heavy chains and twofull-length light chains, but in some instances can include fewer chainssuch as antibodies naturally occurring in camelids, which can compriseonly heavy chains. Antibodies can be derived solely from a singlesource, or can be “chimeric,” that is, different portions of theantibody can be derived from two different antibodies. The antigenbinding proteins, antibodies, or binding fragments can be produced inhybridomas, by recombinant DNA techniques, or by enzymatic or chemicalcleavage of intact antibodies. Unless otherwise indicated, the term“antibody” includes, in addition to antibodies comprising twofull-length heavy chains and two full-length light chains, derivatives,variants, fragments, and muteins thereof. Furthermore, unless explicitlyexcluded, antibodies include monoclonal antibodies, bispecificantibodies, minibodies, domain antibodies, synthetic antibodies(sometimes referred to herein as “antibody mimetics”), chimericantibodies, humanized antibodies, human antibodies, antibody fusions(sometimes referred to herein as “antibody conjugates”), and fragmentsthereof.

As used herein, “specific binding” refers to antibody binding to apredetermined antigen. Typically, the antibody binds with dissociationconstant (K_(D)) of 10E7 M or less, and binds to the predeterminedantigen with a K_(D) that is at least two-fold less than its K_(D) forbinding to a non-specific antigen (e.g., albumin, casein) other than thepredetermined antigen or a closely-related antigen.

The term “K_(assoc)” or “K_(a)”, as used herein, is intended to refer tothe association rate of a particular antibody-antigen interaction,whereas the term “K_(dis)” or “K_(d),” as used herein, is intended torefer to the dissociation rate of a particular antibody-antigeninteraction. The term “K_(D)”, as used herein, is intended to refer tothe dissociation constant, which is obtained from the ratio of K_(d) toK_(a) (i.e., K_(d)/K_(a)) and is expressed as a molar concentration (M).

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG) that is encoded by heavy chain constant region genes.

As used herein, the terms “inhibits binding” and “blocks binding” (e.g.,referring to inhibition/blocking of binding of CD32 ligand, e.g., IgG,to CD32) are used interchangeably and encompass both partial andcomplete inhibition/blocking. The inhibition/blocking of IgG to CD32preferably reduces or alters the normal level or type of effector cellfunctions that occurs when IgG binds to CD32 without inhibition orblocking. Inhibition and blocking are also intended to include anymeasurable decrease in the binding affinity of IgG to CD32 when incontact with an anti-CD32 antibody as compared to the ligand not incontact with an anti-CD32 antibody, e.g., the blocking of CD32 ligandsto CD32 by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,99%, or 100%.

An “Fc” region comprises two heavy chain fragments comprising some orall of the constant “CH” domains of an antibody. The two heavy chainfragments are held together by two or more disulfide bonds. The Fcregion may comprise all or part of the hinge region, either with orwithout additional amino acids from the heavy chain constant region. Inother words, the Fc region may optionally comprise one or both of CH2and CH3.

A “Fab′ fragment” comprises one light chain and a portion of one heavychain that contains the VH domain and the CH1 domain and also the regionbetween the CH1 and CH2 domains, such that an interchain disulfide bondcan be formed between the two heavy chains of two Fab′ fragments to forman F(ab′)2 molecule.

The term “vector” means any molecule or entity (e.g., nucleic acid,plasmid, bacteriophage or virus) used to transfer protein-codinginformation into a host cell.

As used herein, the term “thrombocytopenia” refers to a subnormal numberof platelets in the circulating blood (Wintrobe M M et al. Disorders ofPlatelets and Hemostasis. In: Clinical Hematology, Seventh Edition, Lea& Febiger, Philadelphia, 1974). This is typically defined as a plateletcount less than the lower limit of normal (usually taken as 150×109/L).It may also be characterized as a fall in the number of circulatingplatelets. For example, a fall in the platelet count of 30-50% or more,following administration of heparin, may be a symptom of heparin-inducedthrombocytopenia, even if the platelet count does not fall below150×109/L. (Warkentin T E. Clinical presentation of heparin-inducedthrombocytopenia (October 1998) Semin Hematol 35(4 Suppl 5):9-16;discussion 35-6; PubMed ID: 9855179). The platelet count is measured byelectronic counting methods, usually as part of a Complete Blood Count(CBC).

As used herein, the term “thrombosis” refers to the formation of a bloodclot inside a blood vessel (venous or arterial). Typically the bloodclot, or thrombus, would consist of fibrin and blood cells, includingactivated platelets in various proportions.

As used herein, the phrase “IgG-mediated thrombosis” refers tothrombosis where IgG antibody molecules contribute to the formation ofthe thrombus.

The term “patient” and “subject” are used interchangeably herein torefer to a mammal in need of administration of a therapy.

The terms “disease” and “disorder” as used herein are intended also toinclude medical conditions and syndromes regarded as abnormal orindicative of impaired function, as distinguished from normal health bysigns, symptoms, or laboratory-based diagnostics suggesting the presenceof medical diseases or disorders.

“Treating” includes both treating and preventing.

The term “identity” refers to a relationship between the sequences oftwo or more polypeptide molecules or two or more nucleic acid molecules,as determined by aligning and comparing the sequences. “Percentidentity” means the percent of identical residues between the aminoacids or nucleotides in the compared molecules and is calculated basedon the size of the smallest of the molecules being compared. For thesecalculations, gaps in alignments (if any) are preferably addressed by aparticular mathematical model or computer program (i.e., an“algorithm”). Methods that can be used to calculate the identity of thealigned nucleic acids or polypeptides include those described inComputational Molecular Biology, (Lesk, A. M., ed.), 1988, New York:Oxford University Press; Biocomputing Informatics and Genome Projects,(Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysisof Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.),1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysisin Molecular Biology, New York: Academic Press; Sequence AnalysisPrimer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M.Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

The term “chimeric antibody” is intended to refer to antibodies in whichthe variable region sequences are derived from one species and theconstant region sequences from another. For example, an antibody inwhich the heavy and light chain variable region sequences are derivedfrom a mouse antibody and the constant region sequences are derived froma human antibody, might be described as a mouse-human chimeric antibody.

The term “humanized antibody” is intended to refer to antibodies inwhich CDR sequences derived from antibodies from various mammalianspecies, such as a mouse, have been grafted onto human germline variableframework sequences. Additional framework region amino acidmodifications may be introduced.

The term “effector function” refers to the functional ability of the Fcor constant region of the antibody to bind proteins and/or cells of theimmune system and platelets. Typical effector functions of IgGantibodies include the ability to bind complement protein (e.g., C1q),the neonatal receptor (FcRn), or an IgG Fc receptor (FcgammaR) (e.g.,Fcgamma RI, Fcgamma RII, Fcgamma RIII). The effects of being able tobind one or more of the foregoing molecules include, but are not limitedto antigen-dependent cellular cytotoxicity (ADCC), complement-dependentcytotoxicity (CDC), phagocytosis, opsonization, and effector cellmodulation. Abrogation or decrease of effector function may refer toabrogation or decrease in one or more of the biochemical or cellularactivities induced at least in part by binding of Fc to its receptors orto a complement protein or an effector cell, while maintaining theantigen-binding activity of the variable region of the antibody.

As used herein, an “effector-deficient” antibody is defined as anantibody having an Fc region that has been altered so as to reduce oreliminate Fc-binding to CD16, CD32, and/or CD64 type IgG receptors.

The term “antigen” refers to any natural or synthetic substance thatcould bind specifically to an antibody.

The term “specific binding” refers to antibody binding to apredetermined antigen. Typically, the antibody binds with an affinityequilibrium constant stronger than 10⁻⁷ M, and binds to thepredetermined antigen with at least two-fold stronger binding to anon-specific antigen.

As used herein, the term “immune complex” refers to the molecularstructures consisting of one or more antibody molecules specificallybound to one or more antigen molecules.

The term “epitope” refers to a protein determinant capable of specificbinding to, or specific binding by, an antibody.

EXAMPLES Example 1 Effector-Deficient Monoclonal Antibody MDE-8

The inventors have demonstrated that native human MDE-8 mAbs causeinfusion reactions in mice transgenic for its human antigen (i.e.,CD32A). These mice are referred to herein as “CD32A mice.” Observablesigns of IgG-mediated infusion reactions in CD32A mice includehypothermia, rapid or shallow breathing, hunched posture, and locomotordysfunction; observable signs of severe infusion reactions also includeimmobilization, convulsion, apparent loss of consciousness, and(infrequently) fatality.

Altering the effector domain (i.e., Fc domain) of the MDE-8 mAb to aneffector-deficient IgG format eliminated infusion reactions whenadministered to CD32A mice.

Moreover, when effector-deficient MDE-8 mAbs were provided prior tochallenge with immune complexes, the effector-deficient MDE-8 mAbsprevented immune complex-induced infusion reactions, as well asthrombocytopenia, thrombosis, and shock.

Thus, effector-deficient monoclonal MDE-8 antibodies may be used inplace of native MDE-8 antibodies to treat any CD32a mediated disease ordisorder. The reasons include that the effector-deficient MDE-8antibodies will not elicit infusion reactions as observed with nativeMDE-8. Moreover, when administered prophylactically or therapeutically,effector-deficient MDE-8 antibodies may be used to treat and/or preventany disease or disorder caused by IgG immune complexes.

Materials and Methods

Effector-competent and effector-deficient variants of MDE-8 mAbs (inboth IgG1 and IgG2 formats) were injected intravenously (tail vein) intoCD32A mice. Two effector-deficient variants of MDE-8 were assessed inthis study; E269R and N297A. CD32A mice have been previously describedin McKenie et al., 1999 Apr. 1, J Immunol, 162(7):4311-8, PubMed ID:10201963. After MDE-8 mAb injection (100 micrograms), animals weremonitored for 30 minutes for assessment of infusion reactions. Blood wascollected retro-orbitally before and 30 minutes after MDE-8 mAbinjection. Platelets were counted by flow cytometry from this collectedblood. After 3 hours, some animals were injected intravenously with a200 micro-liter bolus of immune complexes (ICs) consisting of 150micro-grams mouse monoclonal anti-human CD40L antibody (clone M90, amurine IgG1 mAb purified by Protein G chromatography from ATCC HB-12055hybridoma-conditioned media) in balanced stoichiometry with its antigen,CD40L trimer (50 micro-grams) (Peprotech #310-02). Thirty minutes afterIC injection, platelets were again counted. Animals were thenimmediately sacrificed (i.e., 30 minutes after M90+CD40L IC injection),and lungs were harvested, processed for H&E staining, and examinedmicroscopically for the presence of thrombi.

Results Effector-Deficient MDE-8 mAbs do not Cause Infusion Reactionsthat are Seen with Effector Competent MDE-8 Antibodies

When injected intravenously into CD32A mice, native human MDE-8 IgG1antibodies cause infusion reactions characterized by hypothermia, asmeasured by core body temperature (FIG. 1; diamonds). Mice injected withnative human MDE-8 IgG1 antibodies also showed signs of severe infusionreactions, including apparent loss of consciousness (data not shown).Mouse IgG receptors have reduced binding to human IgG2 (See, e.g.,Overdijk et al., Crosstalk between human IgG isotypes and murineeffector cells, 2012 Oct. 1, J Immunol, 189(7): 3430-8, PubMed ID:22956577), which is consistent with the failure of native anti-humanMDE-8 antibodies in IgG2 format to cause hypothermia (FIG. 1; squares).Importantly, two representative effector-deficient human MDE-8 mAbs (inboth IgG1 and IgG2 formats) (antibodies comprising the amino acids ofSEQ ID NO: 69 together with SEQ ID NO: 65 or SEQ ID NO: 67) did notcause infusion reactions as observed with native human MDE-8 IgG1antibodies (FIG. 1; triangles and X's). The failure of the IgG2effector-competent mAb to cause hypothermia (as may be expected), eventhough it did cause thrombocytopenia, may be surprising to one skilledin the art. This surprising finding also makes clear that lack ofhyperthermia does not indicate that the mAb is safe. Theeffector-deficient results provided in this experiment demonstrate thatthe effector-deficient antibodies described herein solve the previouslyunrecognized safety problem of thrombocytopenia.

It was next observed that severe thrombocytopenia followed intravenousinjection of native human MDE-8 mAbs into CD32A transgenic mice in bothIgG1 and IgG2 formats (FIG. 2, columns 1 and 2). In contrast, tworepresentative effector-deficient human MDE-8 mAbs (antibodiescomprising the amino acids of SEQ ID NO: 69 together with SEQ ID NO: 65or SEQ ID NO: 67) did not induce thrombocytopenia when injected intoCD32A mice (FIG. 2, columns 3 and 4). The small reduction in circulatingplatelet numbers seen in FIG. 2 columns 3 and 4 is typical and caused byrepeated blood draws.

These experiments demonstrate that thrombocytopenia is independent ofhypothermia, and that a drop in platelet count is a more sensitiveindicator of infusion reaction than temperature drop, since MDE-8 inIgG2 format failed to cause hypothermia (see FIG. 1) yet largelydepleted circulating platelets (FIG. 2).

Flow cytometric analysis of whole blood from CD32A transgenic micebefore (FIG. 3A) and after (FIG. 3B) intravenous injection of nativehuman MDE-8 mAbs in human IgG1 format showed severe platelet depletion(FIG. 3B). (Fluorescent beads [1 micro-meter] were included to controlfor blood volume [gate P5]; the upper right quadrant includes red bloodcells and white blood cells [gate P4].)

Importantly, when native human MDE-8 IgG1 mAbs were madeeffector-deficient (antibodies comprising the amino acids of SEQ ID NO:69 together with SEQ ID NO: 65), they failed to clear circulatingplatelets (compare FIG. 3C [before injection] and FIG. 3D [afterinjection of effector-deficient human MDE-8 mAbs] with FIG. 3A and FIG.3B). Thus, effector-deficient human MDE-8 mAbs did not deplete platelets(FIG. 3D). Further, mice injected with effector-deficient human MDE-8mAbs (IgG1 E269R and IgG2 N297A) showed no observable signs of infusionreactions (data not shown).

Similar results were obtained when using a different IgG subclass ofeffector-deficient human MDE-8 mAbs: MDE-8 (antibodies comprising theamino acids of SEQ ID NO: 69 together with SEQ ID NO: 67). Flowcytometric analysis of whole blood from CD32A transgenic mice before(FIG. 3E) and after (FIG. 3F) intravenous injection of native MDE-8 mAbIgG2 showed severe platelet depletion (FIG. 3F). Importantly, when thenative MDE-8 mAb IgG2 was made effector-deficient, it no longer clearedcirculating platelets (compare FIG. 3G [before injection] and FIG. 3H[after injection of effector-deficient human MDE-8 mAb] with FIG. 3E andFIG. 3F).

These results show that two representative IgG subclass types ofeffector-deficient human MDE-8 mAbs did not deplete circulatingplatelets (FIGS. 3D and 3H).

The results shown in FIG. 3 demonstrate that the effector domain ofnative MDE-8 IgG mAbs causes infusion reactions in CD32A mice, and suchinfusion reactions are eliminated by altering the IgG-Fc domain. Thus,ablating an anti-CD32a antibody's capacity to efficiently bind IgGFc-receptors is beneficial in eliminating infusion reactions. RenderingMDE-8 mAbs effector-deficient also abrogates the antibodies' capacity toclear platelets from circulating blood, indicating such clearance isalso mediated by the IgG-Fc domain, which, in the case of MDE-8, isimmobilized on the surface of CD32A transgenic mouse platelets.

Effector-Deficient MDE-8 mAbs Protect CD32A Transgenic Mice from ImmuneComplex-Induced Thrombocytopenia

It was next determined that effector-deficient MDE-8 mAbs protect CD32Atransgenic mice against immune complex-induced thrombocytopenia (drop incirculating platelet count). Three hours prior to immune complexchallenge, CD32A mice were treated with vehicle phosphate bufferedsaline (PBS) or one of two representative effector-deficient human MDE-8mAbs (100 micro-grams): 1) effector-deficient MDE-8 IgG1 E269R (SEQ IDNO: 69 together with SEQ ID NO: 65); or 2) effector-deficient MDE-8 IgG2N297A (SEQ ID NO: 69 together with SEQ ID NO: 67). Mice were challengedwith immune complex (M90+CD40L, total of 200 micro-grams), and wholeblood was collected 30 minutes after challenge. FIG. 4 shows theresults. In mice pre-treated with vehicle control, IC injection resultedin the mice having signs of severe shock (data not shown) and severeplatelet depletion. Animals pre-treated with effector-deficient MDE-8IgG1 E269R did not exhibit signs of IC-dependent infusion reactions orshock (data not shown). Moreover, as shown in FIG. 4B, mice pre-treatedwith effector-deficient MDE-8 IgG1 E269R did not experience IC-inducedthrombocytopenia (i.e., platelets were not cleared by ICs; FIG. 4B). Dueto infusion reactions, it was not possible to similarly test effectorcompetent MDE-8 mAbs in native IgG1 format.

Similar results were obtained with effector-deficient MDE-8 IgG2 N297A.FIG. 4C shows the platelet count from a CD32A mouse pre-treated withvehicle control and following IC injection. FIG. 4D shows the post-ICinjection platelet count of a CD32A mouse pre-treated witheffector-deficient MDE-8 IgG2 N297. The data in FIG. 4 arerepresentative of all animals tested.

FIG. 5 shows a bar graph depicting the drop in circulating plateletsfollowing IC injection into CD32A mice pre-treated with either vehicleor effector-deficient MDE-8 antibodies (pre-treatment at three hoursprior to IC challenge). CD32A mice pre-treated with vehicle (PBS) becameseverely thrombocytopenic (FIGS. 5A and 5B, bars #1), whereas micepre-treated with effector-deficient MDE-8 IgG1 E269R (FIG. 5A) oreffector-deficient MDE-8 IgG2 N297A (FIG. 5B) were largely protectedfrom loss of circulating platelets. Animal #1 in FIG. 5A and FIG. 5B waspre-treated with vehicle (PBS). M90+CD40L immune complexes were injectedintravenously into all animals. Thirty minutes later, blood was drawnand platelets were counted. FIG. 5A shows that effector-deficient MDE-8IgG1 E269R protects mice from immune complex-mediated thrombocytopenia(See FIG. 5A, columns 2, 3, and 4). FIG. 5B shows thateffector-deficient MDE-8 IgG2 N297A protects mice from immunecomplex-mediated thrombocytopenia (See FIG. 5B, columns 2 and 3). Thus,two representative IgG subclasses (IgG1, IgG2) of effector-deficientMDE-8 mAbs protected CD32A transgenic mice from immune complex-inducedthrombocytopenia.

Effector-Deficient MDE-8 Antibodies Protect CD32A Transgenic Mice fromPulmonary Thrombosis Caused by Immune Complexes (ICs)

CD32A transgenic mice were pre-treated with vehicle (PBS) or with 100micro-grams of representative effector-deficient MDE-8 mAbs (SEQ ID NO:69 together with SEQ ID NO: 65 or SEQ ID NO: 67). Three hours later,mice were challenged with M90+CD40L ICs (200 micro-grams). After thirtyminutes, mice were sacrificed and their lungs harvested for analysis.FIG. 6A shows an H&E stained lung section from a mouse pre-treated withvehicle three hours prior to IC challenge. Pervasive occlusive pulmonarythrombi (*) were observed in mice pre-treated with vehicle.Surprisingly, mice pre-treated with effector-deficient MDE-8 IgG1 E269Rexhibited normal lung anatomy without evidence of thrombosis (FIG. 6B).Effector-deficient MDE-8 IgG1 E269R pre-treated mice also showed normalblood vessels having abundant red blood cells in normal (healthy)alveolar tissue (FIG. 6B), as compared to vehicle treated mice (FIG.6A), whose blood vessels were abnormal, with fewer numbers of red bloodcells observed in blood vessels, as well as evidence of inflamedalveolar tissue.

Pulmonary thrombi per field were counted by H&E microscopy of mouselungs following IC challenge. Four mice were injected with M90+CD40L IC.Animal #1 (FIG. 6C, bar 1; vehicle control) showed pervasive pulmonarythrombosis (mean of 20 per field), whereas animals #2-4 (FIG. 6C, bars2-4) that were pre-treated with effector-deficient MDE-8 IgG1 E269Rprior to IC challenge exhibited normal lung anatomy without evidence ofthrombosis. The findings depicted in FIG. 6C also demonstrate thateffector-deficient MDE-8 IgG1 E269R did not cause pulmonary thrombosis.

Similar results were obtained with effector-deficient MDE-8 IgG2 N297A.FIG. 7A shows an H&E stained lung section from a mouse which had beenpre-treated with vehicle three hours prior to IC challenge. In FIG. 7A,pervasive occlusive pulmonary thrombi (*) were observed. In contrast,mice pre-treated with effector-deficient MDE-8 IgG2 N297A exhibitednormal lung anatomy without evidence of thrombosis (FIG. 7B).Effector-deficient MDE-8 IgG2 N297A pre-treated mice also showed normalblood vessels having abundant red blood cells amidst healthy alveolartissue (FIG. 7B), in contrast to the abnormal (with fewer numbers of redblood cells observed in blood vessels, as well as evidence of inflamed)alveolar tissue of the vehicle (PBS) pre-treated mice (FIG. 7A). Thedata in FIGS. 6 and 7 is representative of all animals tested.

FIG. 7C shows H&E microscopy of mouse lungs after IC challenge in micepre-treated with vehicle or effector-deficient MDE-8 antibodies.Pulmonary thrombi per field were counted. Four mice were injected withM90+CD40L IC. Animal #1 (FIG. 7C, bar 1; control) showed pervasivepulmonary thrombosis (mean of 18.6 per field), whereas animals #2 and #3(FIG. 7C, bars 2 and 3), which were pre-treated with effector-deficientMDE-8 IgG2 N297A, exhibited normal lung anatomy without evidence ofthrombosis. These findings also demonstrate that MDE-8 IgG2 N297A mAb byitself did not cause pulmonary thrombosis.

Taken together, the data presented in Example 1 demonstrate: (1) thatnative (effector competent) anti-CD32a IgG mAbs cause infusionsreactions and induce thrombocytopenia; (2) that altering MDE-8 mAbs toan effector-deficient format renders the IgG of choice infusion-safe andhemostatically safe (in that it does not induce thrombocytopenia); (3)that native MDE-8 mAb mediated infusion reactions and thrombocytopeniaare dependent on the function of the IgG-Fc (effector) domain; (4) thateffector-deficient MDE-8 IgG1 and IgG2 mAbs protect CD32A transgenicmice from immune complex-mediated infusion reactions, shock,thrombocytopenia, and thrombosis; and (5) that the CD32A IgG receptorlargely controls infusion reactions, thrombocytopenia, thrombosis, andshock as mediated by ICs in these immunologically intact (e.g., havingthe full array of murine IgG receptors) CD32A transgenic mice. Thedominant effect of the human CD32A transgene product over all othermouse IgG-Fc receptors (murine FcgammaRT, FcgammaRIIb, and FcgammaRIII),in response to thrombotic ICs, is an unexpected finding.

Example 2 Effector-Deficient Chimeric Monoclonal Antibody AT-10

The inventors have further demonstrated that native chimeric(mouse-human) AT-10 IgG mAbs cause infusion reactions in mice transgenicfor CD32a (e.g., in CD32A mice). Observable signs of IgG-mediatedinfusion reactions in CD32A mice include hypothermia, rapid or shallowbreathing, hunched posture, and locomotor dysfunction. Observable signsof severe infusion reactions include immobilization, convulsion, and(infrequently) fatality.

We show herein that altering the effector domain (i.e., the Fc domain)of the native AT-10 mAbs to an effector-deficient IgG format eliminatedinfusion reactions when administered to CD32A mice.

Moreover, when effector-deficient AT-10 mAbs were administered prior tochallenge with immune complexes, the effector-deficient AT-10 mAbsprevented immune complex-induced infusion reactions, as well asthrombocytopenia and thrombotic shock.

Thus, effector-deficient monoclonal AT-10 antibodies may be used inplace of native AT-10 antibodies to treat any CD32a mediated disease ordisorder. The reasons include that the effector-deficient AT-10antibodies will not elicit infusion reactions as observed with nativeAT-10 mAbs. Moreover, effector-deficient AT-10 mAbs may be used to treatand/or prevent any disease or disorder caused by immune complexes whengiven prophylactically or therapeutically.

Materials and Methods

Effector-competent and effector-deficient variants of AT-10 mAbs (IgG1and IgG1 E269R, respectively) were injected intravenously (tail vein)into CD32A mice (as in Example 1). After injection (100 micro-grams),mice were monitored for 30 minutes for assessment of infusion reactions.Blood was collected (retro-orbitally) before, and 30 minutes after AT-10mAb injection. Platelets were counted by flow cytometry from thiscollected blood. After 3 hours, some animals were injected with immunecomplexes (ICs, as in Example 1, 200 micro-grams), and blood wascollected 30 minutes after injection. Platelets were again counted fromthis collected blood. Lungs were harvested 30 minutes after injection ofICs, processed for H&E staining, and examined microscopically for thepresence of thrombi.

Results Effector-Deficient AT-10 mAbs do not Cause Infusion Reactionsthat are Seen with Effector Competent AT-10 Antibodies

When injected intravenously into CD32A transgenic mice, native chimericAT-10 mAbs cause infusion reactions characterized by thrombocytopenia(FIG. 8, bar 1). Notably, these mice did not have hypothermia data notshown). Thrombocytopenia was ablated when effector-deficient chimericAT-10 human IgG1 E269R mAbs were administered in lieu of native chimericAT-10 human IgG1 (FIG. 8, column 2) (antibodies comprising the aminoacids of SEQ ID NO: 22 together with SEQ ID NO: 16).

Flow cytometric analysis of whole blood from CD32A mice before (FIG. 9A)and after (FIG. 9B) intravenous injection of native chimeric AT-10 humanIgG1 showed severe platelet depletion (FIG. 9B) despite having nohypothermia. Importantly, when native chimeric AT-10 human IgG1 mAbswere made effector-deficient, they no longer reduced circulatingplatelets (compare FIG. 9C [before injection] and FIG. 9D [afterinjection of effector-deficient chimeric AT-10 human IgG1 mAbs]). Thus,effector-deficient AT-10 mAbs did not deplete platelets. This datademonstrates that the IgG effector domain is responsible for AT-10IgG-induced thrombocytopenia.

These experiments demonstrate that thrombocytopenia (a drop in plateletcount) is independent of and, in this case, a more sensitive indicatorof infusion reaction than temperature drop, since native AT-10 mAbs inIgG1 format failed to cause hypothermia (data not shown) but causedsevere platelet depletion. Importantly, effector-deficient AT-10 mAbsdid not deplete platelets (i.e., cause thrombocytopenia) like theireffector competent counterparts.

The results shown in FIGS. 8 and 9 demonstrate that the effector regionof native AT-10 IgG mAbs mediates infusion reactions in CD32A mice, andthat such infusion reactions are eliminated by altering the IgG-Fcregion. Thus, ablating an anti-CD32a antibody's capacity to efficientlybind IgG Fc-receptors is beneficial in eliminating infusion reactions.

Effector-Deficient AT-10 mAbs Protect CD32A Transgenic Mice from ImmuneComplex-Induced Thrombocytopenia

Next it was demonstrated that effector-deficient AT-10 mAbs were capableof protecting CD32A transgenic mice from immune complex-inducedthrombocytopenia (FIG. 10). Three hours prior to immune complexchallenge, mice were treated with PBS vehicle or an effector-deficientAT-10 mAb (IgG1 E269R,) (antibodies comprising the amino acid of SEQ IDNO: 22 together with SEQ ID NO: 16). Immune complexes (M90+CD40L (as inExample 1); 200 micro-grams) were injected intravenously and whole bloodwas collected 30 minutes after IC challenge. Platelets were counted fromthis collected blood.

FIG. 10 shows a bar graph depicting the % drop in circulating plateletsfollowing IC injection into CD32A mice pre-treated with either vehicleor effector-deficient chimeric AT-10 human IgG1 E269R antibodies. CD32Amice pre-treated with vehicle (PBS) became severely thrombocytopenic(FIG. 10 column 1), whereas mice pre-treated with effector-deficientchimeric AT-10 human IgG1 E269R (FIG. 10 columns 2 and 3) were largelyprotected from loss of circulating platelets. Effector-deficientchimeric AT-10 human IgG1 E269R protected mice from immunecomplex-induced thrombocytopenia (FIG. 10, columns 2 and 3 compared tocontrol, column 1.

FIG. 11 shows a flow cytometric analysis of circulating plateletsfollowing IC injection into CD32A mice pre-treated with either vehicleor effector-deficient AT-10 antibodies as described above. FIG. 11Ashows platelets depletion from a mouse that received vehicle. FIG. 11Bshows that animals pre-treated with effector-deficient chimeric AT-10human IgG1 E269R did not experience thrombocytopenia in response to ICinjection. Furthermore, effector-deficient chimeric AT-10 human IgG1E269R pre-treatment completely protected CD32A mice from observablesigns of IC-mediated infusion reaction. Chimeric AT-10 human IgG1E269R-treated animals appeared unaffected by IC injection, whereasvehicle treated controls showed signs of imparied mobility, hunchedposture, and shallow and rapid breathing, consistent with shock.

Effector-Deficient AT-10 Antibodies Protect CD32A Transgenic Mice fromPulmonary Thrombosis Caused by Immune Complexes (ICs)

Mice were pre-treated with vehicle (PBS) or with effector-deficientAT-10 mAbs (antibodies comprising the amino acids of SEQ ID NO: 22together with SEQ ID NO: 16). Three hours later, mice were challengedwith M90+CD40L IC (as in Example 1; 200 micro-grams). After thirtyminutes, mice were sacrificed and their lungs removed for analysis. FIG.12A shows a representative H&E stained lung section from a mousepre-treated with vehicle 3 hours prior to IC challenge. Pervasiveocclusive pulmonary thrombi (*) were detected in vehicle-treated mice.Surprisingly, mice pre-treated with effector-deficient AT-10 IgG1 E269Rexhibited normal lung anatomy without evidence of thrombosis (FIG. 12B),despite the fact that these mice are immunologically intact (i.e., havethe full repertoire of normal mouse IgG receptors, in addition to humanCD32A). Effector-deficient AT-10 IgG1 E269R pre-treated mice also showednormal blood vessels having abundant red blood cells amidst healthyalveolar tissue (FIG. 12B), as compared to control (vehicle-treated)mice (FIG. 12A). These results demonstrate that effector-deficientchimeric AT-10 human IgG1 E269R mAbs are capable of protecting againstIC-induced thrombosis.

FIG. 12C shows H&E microscopy of mouse lungs following IC injection intoCD32A mice pre-treated with either vehicle (control) oreffector-deficient chimeric AT-10 antibodies as described above (SeeFIGS. 12A and 12B). Animal #1 (FIG. 12C, bar 1; control) showedpervasive pulmonary thrombosis (mean of 17.6 clots per field), whereasanimals #2 and #3 (FIG. 12C, bars 2 and 3), which were pre-treated witheffector-deficient chimeric AT-10 human IgG1 E269R, exhibited normallung anatomy without evidence of thrombosis.

Humanized Effector-Deficient AT-10 mAbs

An effector-deficient humanized AT-10 IgG1 E269R mAb (“hAT-10”) was madeand tested (antibodies comprising the amino acids of SEQ ID NO: 24together with SEQ ID NO: 20). In FIG. 13A, hAT-10 E269R mAb (116 μg) wasadministered to CD32A mice and core body temperature of the mice wasassessed over time. hAT-10 E269R mAbs did not cause hypothermia (anindicator of infusion reaction). In addition to not exhibitinghypothermia, these animals exhibited no other signs of having infusionreactions. FIG. 13B shows the results of a study where platelets fromCD32A mice were assessed before and after hAT-10 E269R injection (116micro-grams). hAT-10 had no effect on circulating platelet counts(compare FIG. 13B with FIG. 13C).

Furthermore, hAT-10 E269R completely protects mice against M90+CD40LIC-induced thrombocytopenia. Control animals that received vehicle (PBScontrol) pre-treatment became unconscious within 10 minutes of receivingIC challenge and subsequently showed signs consistent with severe shock.In contrast, mice pre-treated with hAT-10 E269R appeared unaffected byIC challenge (data not shown). hAT-10 E269R pre-treatment also protectedCD32A mice from thrombocytopenia (compare FIG. 13D showing platelet lossin vehicle-treated group and FIG. 13E showing no platelet loss in hAT-10E269R-treated animals).

Finally, humanized effector-deficient AT-10 IgG1 E269R (hAT-10 E269R)antibody protected mice from immune complex-induced pulmonarythrombosis. As observed with AT-10 chimeric antibody (FIG. 12),pre-treatment with hAT-10 E269R completely prevented pulmonarythrombosis in CD32A mice after IC challenge (M90+CD40L; as in Example 1;200 micro-grams). FIG. 13F shows significant thrombi in lungs of controltreated mice and nearly complete lack of thrombi in hAT-10 E269R treatedmice. See, also, FIG. 13G showing pervasive pulmonary thrombosis (meanof 24.4 clots per field) in control treated mice as compared to a lackof thrombi in mice pre-treated with hAT-10 E269R (FIG. 13H).

Taken together, the data presented in Example 2 demonstrate: (1) thatnative (effector competent) anti-CD32a IgG mAb AT-10 causes infusionsreactions characterized by thrombocytopenia; (2) that altering AT-10 mAbto be effector-deficient renders AT-10 infusion-safe and hemostaticallysafe (in that it does not induce thrombocytopenia); (3) that native(effector competent) AT-10 antibody mediated infusion reactions andthrombocytopenia are dependent on the function of the IgG-Fc (effector)domain; (4) that effector-deficient AT-10 IgG mAb protects CD32Atransgenic mice from immune complex-mediated infusion reactions,thrombocytopenia, and thrombosis; and (5) that the CD32a IgG receptorlargely controls infusion reactions, thrombocytopenia, thrombosis, andshock, as mediated by ICs in these immunologically intact CD32A mice.

Example 3 Effector-Deficient Chimeric Monoclonal Antibody IV.3

The inventors have further demonstrated that native chimeric IV.3 mAbscause infusion reactions in mice transgenic for its antigen (i.e., CD32Amice). Observable signs of IgG-mediated infusion reactions in CD32A miceinclude hypothermia, rapid or shallow breathing, hunched posture, andlocomotor dysfunction; observable signs of severe infusion reactionsalso include impaired mobility, convulsion, apparent loss ofconsciousness, and (infrequently) fatality.

We show herein that altering the effector domain (i.e., Fc domain) ofchimeric IV.3 to an effector-deficient IgG format eliminated infusionreactions following administration to CD32A mice.

Moreover, when effector-deficient IV.3 was provided to subjects prior tochallenge with immune complexes, the effector-deficient IV.3 mAbsprevented immune complex-induced infusion reactions, as well asthrombocytopenia and thrombotic shock.

Thus, effector-deficient monoclonal IV.3 antibodies should be used inplace of native IV.3 antibodies to treat any CD32a mediated disease ordisorder. The reasons include that the effector-deficient IV.3antibodies will not elicit infusion reactions as observed with nativeIV.3 mAbs. Moreover, effector-deficient IV.3 may be used to treat and/orprevent any disease or disorder caused by immune complexes when givenprophylactically or therapeutically.

Materials and Methods

In this set of experiments, effector-competent and effector-deficientvariants of chimeric IV.3 mAbs (human IgG2 and human IgG2 N297A,respectively) were injected intravenously (tail vein) into human CD32Atransgenic mice (as in Example 1). After injection (100 micro-grams),animals were monitored for 30 minutes for assessment of infusionreactions. Blood was collected (retro-orbitally) before and 30 minutesafter IV.3 mAb injection. Platelets were then counted by flow cytometryfrom this collected blood. After 3 hours, some animals were injectedwith immune complexes (ICs, as in Example 1, 200 micro-grams), afterwhich (30 minutes) platelets were again counted. Lungs were thenharvested, processed for H&E staining, and examined microscopically forthe presence of thrombi.

Results Effector-Deficient IV.3 mAbs do not Cause Infusion Reactionsthat are Seen with Effector Competent IV.3 Antibodies

We discovered that, when injected intravenously into CD32A transgenicmice, effector competent chimeric IV.3 human IgG2 mAb causes infusionreactions in a dose-dependent manner, as characterized bythrombocytopenia without hypothermia. FIG. 14 shows that, followingintravenous injection of chimeric IV.3 in native human IgG2 format,CD32A transgenic mice became severely thrombocytopenic (FIG. 14A, solidbars). This thrombocytopenia was largely ablated when effector-deficientchimeric IV.3 human IgG2 N297A mAb (antibodies comprising the aminoacids of SEQ ID NO: 51 together with SEQ ID NO: 45) was administered inlieu of native chimeric IV.3 (FIG. 14A, open bars). FIG. 14B shows thatneither native chimeric IV.3 human IgG2 nor its effector-deficient (IV.3IgG2 N297A) format caused hypothermia in CD32A transgenic mice.

These results again suggest that a drop in platelet count is independentof and, in this case, more sensitive than core body temperature drop asindicator for infusion reaction to antibodies that interact withplatelets in vivo.

Flow cytometric analysis of whole blood from CD32A transgenic micebefore (FIG. 15A) and after (FIG. 15B) intravenous injection of nativechimeric IV.3 in human IgG2 showed severe platelet depletion (FIG. 15B).Importantly, when native chimeric IV.3 IgG2 mAb was madeeffector-deficient, it no longer reduced circulating platelets (compareFIG. 15C [before injection] and FIG. 15D [after injection ofeffector-deficient chimeric IV.3 mAbs]). Thus, effector-deficient IV.3mAbs did not deplete platelets (FIG. 15D). This data demonstrates thatthe IgG effector domain is responsible for the IV.3 IgG-inducedthrombocytopenia.

The results shown in FIGS. 14 and 15 demonstrate that the effectordomain of native IV.3 IgG mAbs cause infusion reactions in CD32A mice,and such infusion reactions are eliminated by altering the IgG-Fcdomain. Thus, ablating an anti-CD32a antibody's capacity to efficientlybind IgG Fc-receptors is beneficial in eliminating infusion reactions.Rendering IV.3 mAbs effector-deficient also abrogates the antibodies'capacity to clear platelets from circulating blood, indicating suchclearance is also mediated by the IgG-Fc domain, which, in the case ofIV.3, is immobilized on the surface of CD32A transgenic mouse platelets.

Effector-Deficient IV.3 mAbs Protect CD32A Transgenic Mice from ImmuneComplex-Induced Thrombocytopenia

It was next determined that effector-deficient chimeric IV.3 mAbsprotect CD32A transgenic mice from immune complex-inducedthrombocytopenia. Three hours prior to immune complex challenge, CD32Atransgenic mice were pre-treated with PBS vehicle or 100 micro-grams ofa representative effector-deficient chimeric IV.3 IgG2 N297A (antibodiescomprising the amino acids of SEQ ID NO: 51 together with SEQ ID NO:45). Whole blood was collected 30 minutes after mice were challengedwith immune complexes (M90+CD40L, as in Example 1, 200 micro-grams).FIG. 16A shows platelet depletion in a whole blood sample from a mousethat received vehicle. FIG. 16B shows that animals pre-treated withchimeric IV.3 IgG2 N297A did not experience thrombocytopenia in responseto ICs.

FIG. 17 shows a bar graph depicting the % drop in circulating plateletsfollowing IC injection (M90+CD40L, as in Example 1, 200 micro-grams)into CD32A mice pre-treated with either vehicle or effector-deficientchimeric IV.3 antibodies as described above. CD32A mice pre-treated withvehicle (PBS) became severely thrombocytopenic (FIG. 17, bar 1), whereasmice pre-treated with effector-deficient chimeric IV.3 IgG2 N297A (FIG.17, bars 2, 3 and 4) were largely protected from loss of circulatingplatelets. Thus, effector-deficient chimeric IV.3 human IgG2 N297Aprotected mice from immune complex-induced thrombocytopenia (FIG. 17).

Effector-Deficient Chimeric IV.3 Antibodies Protect CD32A TransgenicMice from Pulmonary Thrombosis Caused by Immune Complexes (ICs)

In this experiment, CD32A mice were pre-treated with vehicle or with 100micro-grams of effector-deficient chimeric IV.3 human IgG2 N297A mAb(SEQ ID NO: 51 together with SEQ ID NO: 45). Three hours later,pre-treated mice were challenged with M90+CD40L IC (as in Example 1, 200micro-grams). Thirty minutes later, mice were sacrificed and their lungsremoved for analysis. FIG. 18A shows an H&E stained lung section from amouse pre-treated with vehicle 3 hours prior to IC challenge. Pervasiveocclusive pulmonary thrombi (*) were detected in vehicle-treated mice.Surprisingly, mice pre-treated with effector-deficient chimeric IV.3human IgG2 N297A exhibited normal lung anatomy without evidence ofthrombosis (FIG. 18B). Chimeric IV.3 IgG2 human N297A-pre-treated micealso showed normal blood vessels having abundant red blood cells amidsthealthy alveolar tissue (FIG. 18B), as compared to vehicle treated mice(FIG. 18A), which exhibited abnormal and occluded blood vessels.Notably, these mice were immunologically intact (having the full arrayof naturally occurring mouse IgG-Fc receptors), demonstrating thedominance of CD32a in IC-induced thrombocytopenia, thrombosis, andshock.

In FIG. 18C, the average number of pulmonary thrombi per 10 fields wasdetermined by H&E microscopy of mouse lungs following IC challenge. Fourmice were injected with M90+CD40L IC as described above. Animal #1 (PBSpre-treated control) showed pervasive pulmonary thrombosis (mean of 17.6clots per field), whereas animals #2, #3, and #4 which were pre-treatedwith effector-deficient chimeric IV.3 human IgG2 N297A, exhibited normallung anatomy without evidence of thrombosis.

Taken together, the data presented in Example 3 demonstrate: (1) thatnative chimeric IV.3 anti-CD32a IgG2 mAb causes infusions reactions andinduces thrombocytopenia; (2) that altering chimeric IV.3 mAb to aneffector-deficient format renders chimeric IV.3 infusion-safe andhemostatically safe (in that it does not induce thrombocytopenia); (3)that IV.3 mediated infusion reactions and thrombocytopenia are dependenton the function of the IgG-Fc (effector) domain; (4) thateffector-deficient chimeric IV.3 IgG mAbs protects CD32A transgenic micefrom immune complex-mediated infusion reactions, thrombocytopenia,thrombosis, and shock.

Example 4

Anti-CD32a mAbs potently inhibit CD32a-mediated immune complex-inducedhuman platelet aggregation and degranulation.

In this example we analyzed immune complex-induced human plateletaggregation and degranulation in vitro to assess the potency andefficacy of anti-CD32a mAbs.

Methods

Platelet-activating immune complexes (ICs) were prepared by combiningCD40 ligand (CD40L, also called CD154), human platelet factor 4 (hPF4),human beta 2-Glycoprotein I (beta 2-GPI), or TNFalpha antibodies withtheir respective ligands typically at balanced stoichiometry (100-1000nM). The following types of immune complexes were tested: (1) M90anti-CD40L mAb+CD40L; (2) M91 anti-CD40L mAb+CD40L; (3) M90 anti-CD40LmAb+M91 anti-CD40L mAb+CD40L (a polyclonal immune complex); (4)anti-hPF4 mAb+hPF4+0.1 U/ml heparin (an HIT-like IC); (5) polyclonalanti-beta 2-GPI+beta 2-GPI (an APS-like IC); (6) infliximab+TNFalpha (atherapeutic mAb-like IC); (7) adalimumab+TNFalpha (a therapeuticmAb-like IC); and (8) goat F(ab′)2-anti-human-IgG-F(ab′)2+infliximab (tomimic anti-therapeutic antibody IC activity).

Isolated platelets were assessed via light-transmission aggregometry asfollows. Platelets were acquired from healthy human donors (n=10)following informed consent, washed and suspended in assay buffer.Platelets were placed in cuvettes in the aggregometer and allowed toincubate at 37° C. until a stable baseline was achieved.

Anti-CD32a antibodies or saline were added to the cuvette 5-10 minutesbefore the addition of platelet-activating immune complexes. Followingthe addition of immune complexes, aggregation traces were monitored forat least 5 minutes. In some cases where CD32a mAbs prevented immunecomplex-induced aggregation, the capacity of the platelets to aggregatewas confirmed by the addition of the standard agonist collagen (7micro-grams/milliliter final concentration).

Results

In FIG. 19, 500 nM M90+CD40L IC activated CD32a on washed humanplatelets, leading to aggregation, which was not blocked by a controlmAb (recombinant rabbit IgG) FIG. 19 (line 1). Platelet aggregation wascompletely blocked by mouse IV.3 mIgG2b (FIG. 19, line 2) and by nativechimeric IV.3 hIgG1 (FIG. 19, line 3), which display similar potency (<5nM required). This data demonstrates that cloned native chimeric IV.3hIgG1 has CD32a blocking activity comparable to that of the parent mousemonoclonal antibody.

In FIG. 20, 250 nM M90+CD40L IC potently induced CD32a-dependentaggregation (FIG. 20, line 1), which was blocked by 7 nM aglycosylatedmouse IV.3 mIgG2b (FIG. 20, line 2). These results suggest thatdeglycosylation of the “Fc” effector domain of mouse IV.3 mIgG2b mAb,which renders the antibody effector-deficient, does not significantlyalter its potency in blocking platelet CD32a (i.e., its Fab-dependentactivity).

In FIG. 21, 500 nM M90+CD40L IC induced aggregation (FIG. 21, line 1),which was blocked by native chimeric IV.3 hIgG2 (FIG. 21, lines 2, 3,4). Aggregation of platelets from a second donor tested with 500 nMM91+CD40L IC was also completely inhibited by native chimeric IV.3 hIgG2(data not shown).

In FIG. 22, 500 nM M90+CD40L IC-induced aggregation (FIG. 22, line 1) isblocked by 3.3 nM (FIG. 22, line 2) and by 1.7 nM (FIG. 22, line 3)effector-deficient chimeric IV.3 hIgG2 N297A mAb (SEQ ID NO: 51 togetherwith SEQ ID NO: 45). Collagen (designated by * here and below), added at19 min, demonstrated the anti-CD32a mAb-treated platelets remainedaggregation competent.

In FIG. 23, 500 nM M90+CD40L IC induced aggregation (FIG. 23, line 1) isblocked by 13 nM effector-deficient chimeric AT-10 hIgG1 E269R (FIG. 23,line 2) mAb (SEQ ID NO: 22 together with SEQ ID NO; 16). Also, nativechimeric AT-10 hIgG1, hIgG2, and effector-deficient chimeric hIgG2 N297Aformats (SEQ ID NO: 22 together with SEQ ID NO: 18) gave similar resultswith platelets from other donors (data not shown).

In FIG. 24, an IC composed of a 700 nM mix of M90+M91 plus CD40L causedplatelet aggregation (FIG. 24, line 1). The activity of this IC wascompletely blocked by less than 3 nM of effector-deficient human MDE-8IgG1 E269R (SEQ ID NO: 69 together with SEQ ID NO: 65) (FIG. 24, line2). Collagen, added at 15 min, demonstrated aggregation competence (FIG.24, line 2*). Native human MDE-8 IgG1, IgG2, and effector-deficient IgG2N297A (SEQ ID NO: 69 together with SEQ ID NO: 67) similarly blockedIC-induced aggregation (data not shown).

In FIG. 25, 500 nM M90+CD40L IC-induced aggregation (FIG. 25, line 1)was inhibited similarly by 3 nM effector-deficient humanized IV.3.1 IgG1E269R (SEQ ID NO: 53 together with SEQ ID NO: 47, FIG. 25, line 2) andby 3 nM mouse IV.3 mIgG2b (FIG. 25, line 3) mAbs. An identicalconcentration (3 nM) of effector-deficient humanized IV.3.2 IgG1 E269RmAbs (SEQ ID NO: 53 together with SEQ ID NO: 49) also inhibited theactivity of this IC (data not shown). This data demonstrates similarpotency between humanized and mouse IV.3 mAbs.

In FIG. 26, 500 nM M90+CD40L IC-induced aggregation (FIG. 26, line 1)was not inhibited by 2 nM effector-deficient humanized IV.3.1 IgG1 E269RmAb (SEQ ID NO: 53 together with SEQ ID NO: 47, FIG. 26, line 2) but wasinhibited by 2 nM mouse IV.3 mIgG2b mAb (FIG. 26, line 3). Collagen,added at 10.5 min, demonstrated platelet aggregation competence.

In FIG. 27, M90+CD40L (500 nM) IC-induced aggregation (FIG. 27, line 1)was blocked by 6 nM effector-deficient humanized IV.3.1 IgG1 E269R mAb(SEQ ID NO: 53 together with SEQ ID NO: 47, FIG. 27, line 2) Collagen(*) was added (FIG. 27, line 2) at 11 min and demonstrated plateletaggregation competence.

In FIG. 28, an IC composed of a 1000 nM mix of M90+M91 plus CD40L causedplatelet aggregation (FIG. 28, line 1). The activity of this IC wasblocked by 25 nM effector-deficient humanized IV.3.1 IgG1 E269R mAb (SEQID NO: 53 together with SEQ ID NO: 47, FIG. 28, line 2) and by 25 nMmouse IV.3 mIgG2b (FIG. 28, line 3). Negative control (no IC added; FIG.28, line 4) did not aggregate.

In FIG. 29, 250 nM hPF4+anti-hPF4+heparin (0.1 Units/milliliter), anHIT-like IC (FIG. 29, line 1), was completely blocked by 40 nM ofeffector-deficient humanized IV.3.1 IgG1 E269R (SEQ ID NO: 53 togetherwith SEQ ID NO: 47, FIG. 29, line 2). Collagen was added (FIG. 29, line2) at 10.5 min. Effector-deficient humanized IV.3.2 IgG1 E269R mAbs (SEQID NO: 53 together with SEQ ID NO: 49) also inhibited the activity ofthis IC (data not shown).

In FIG. 30, 500 nM anti-human beta 2-GPI polyclonal antibody+125 nMhuman beta 2-GPI IC (an APS-like IC) induced robust platelet aggregation(FIG. 30, line 1) which was blocked both by 33 nM of effector-deficienthumanized IV.3.1 IgG1 E269R mAb (SEQ ID NO: 53 together with SEQ ID NO:47, FIG. 30, line 2) and by 50 nM of effector-deficient human MDE-8 IgG1E269R (SEQ ID NO: 69 together with SEQ ID NO: 65, FIG. 30, line 3).

In FIG. 31, M90+CD40L (500 nM) IC-induced aggregation (FIG. 31, line 1)was inhibited by 15 nM effector-deficient humanized AT-10 IgG1 E269R mAb(SEQ ID NO: 24 together with SEQ ID NO: 20, FIG. 31, line 2). Additionof 375 nM of the F(ab′)2 fragment of goat anti-human-F(ab′)2 (designatedas #; added at 16 min), which lacks an Fc-domain, cause plateletaggregation thus demonstrating platelet surface localization ofeffector-deficient humanized AT-10 IgG1 E269R as well as aggregationcompetence.

Taken together, the results depicted by FIG. 19-31 demonstrate thatseveral different types of immune complexes (ICs) are capable ofinducing platelet aggregation in a CD32a-dependent manner. These ICs arepotently blocked by chimeric or humanized IV.3, by chimeric or humanizedAT-10, and by MDE-8, including IgG1 and IgG2 isotype subclasses, in botheffector-competent and effector-deficient formats. Because humanizedAT-10 and humanized IV.3 mAbs exhibited CD32a blocking activitycomparable to that of their parent murine mAbs, these previouslyundescribed and novel mAbs may be useful for treatment of humandisorders in which immune complexes play a pathologic role via CD32a.

When considered together, the in vivo (mouse) and in vitro (aggregation,degranulation) data also demonstrate that effector-deficient formats ofIV.3, AT-10, and MDE-8, whether in IgG1 or IgG2 format, and whetherchimeric, humanized, or fully human, can be expected to have safe invivo administration profiles while providing potent blockade of CD32a,thus preventing CD32a activation induced by ICs or by immobilized IgG.

Combined AT-10, IV.3 and MDE-8 mAbs do not Activate CD32a.

We next considered whether the effector-deficient chimeric, humanized,and human anti-CD32a mAbs described herein were capable, when combined,of activating CD32a (i.e., by directly multimerizing or clustering thereceptor). To this end, we combined 2 nM humanized AT-10 (SEQ ID NO: 24together with SEQ ID NO: 20), 150 nM humanized IV.3.1 (SEQ ID NO: 53together with SEQ ID NO: 47), and 150 nM human MDE-8 (SEQ ID NO: 69together with SEQ ID NO: 65), all in effector-deficient IgG1 E269Rformat, and exposed the combination of these anti-CD32a mAbs to washedhuman platelets. FIG. 32 shows that 500 nM M90+CD40L IC induced robustplatelet aggregation (FIG. 32, line 1), while combined anti-CD32a mAbsdid not cause aggregation (FIG. 32, line 2). Collagen (*), added at 18min, demonstrated aggregation competence. FIG. 33 shows that thecombination of 300 nM effector-deficient chimeric AT-10 hIgG1 E269R (SEQID NO: 22 together with SEQ ID NO: 16), 300 nM effector-deficientchimeric IV.3 hIgG2 N297A (SEQ ID NO: 51 together with SEQ ID NO: 45),and 300 nM effector-deficient human MDE-8 IgG1 E269R (SEQ ID NO: 69together with SEQ ID NO: 65) also failed to induce platelet aggregation,whereas collagen (*) succeeded.

We next examined the capacity of combined CD32a mAbs to induce plateletdegranulation, as occurs when CD32a is clustered by ICs. We alsoevaluated the capacity of these anti-CD32a mAbs to prevent plateletdegranulation caused by therapeutic TNFalpha antibodies complexed withTNFalpha (100 nM). To that end, we tested murine IV.3 mIgG2b,effector-deficient humanized IV.3.1 hIgG1 E269R (SEQ ID NO: 53 togetherwith SEQ ID NO: 47), effector-deficient chimeric AT-10 hIgG1 E269R (SEQID NO: 22 together with SEQ ID NO: 16, and effector-deficient humanMDE-8 IgG1 E269R (SEQ ID NO: 69 together with SEQ ID NO: 65), as well asthe combination of these four mAbs (all at 100 nM), for their capacityto activate washed human platelets, as measured by degranulation in theserotonin release assay (FIG. 34). The results showed that all testedanti-CD32a mAbs prevented TNFalpha-immune-complex induced plateletdegranulation, and that the combination of all mAbs failed todegranulate platelets. FIG. 34.

Further, a combination of 25 nM humanized IV.3.1 (SEQ ID NO: 53 togetherwith SEQ ID NO: 47), 90 nM humanized AT-10 (SEQ ID NO: 24 together withSEQ ID NO: 20), and 75 nM human MDE-8 (SEQ ID NO: 69 together with SEQID NO: 65), all in effector-deficient IgG1 E269R format, was alsosimilarly tested with the same result (i.e., no activation of CD32a;data not shown).

Each of the tested mAbs blocked infliximab and adalimumab anti-TNFalphaIC-induced platelet activation. The combination of four anti-CD32a mAbs(mouse IV.3, humanized IV.3, chimeric AT-10, and human MDE-8) failed tocause any platelet activation (FIG. 34).

Taken together, the results shown in FIGS. 32-34 indicate that thetested mAbs are compatible in that they do not synergistically clusterCD32a to the point of activation, suggesting that these mAbs couldpotentially be delivered to patients without synergistic activation.Furthermore, the compatability of these mAbs provides a progressivetreatment course for anti-CD32a therapy, wherein patients developingimmune reactivity to a first therapeutic anti-CD32a mAb are providedwith follow-up therapeutics compatible for long term treatment ofchronic immune complex-mediated disorders.

Although humanized and human antibodies used for therapy in patientswith immune complex-mediated disorders are expected to be lessimmunogenic, the evidence suggests that many such patients neverthelessdevelop immune reactions to the therapeutic antibody (e.g.,anti-therapeutic-antibody-antibodies, or ATA). These host responseantibodies can form immune complexes that activate CD32a; however, theeffector-deficient CD32a mAbs described herein are candidates for use inprotecting patients from these ATA immune complexes.

Following this rationale, we examined the capacity of anti-CD32a mAbs toprevent platelet degranulation induced by ICs formed from infliximab andF(ab′)2 fragments of goat anti-human IgG-F(ab′)2 antibodies, wherein theonly functional Fc-domain of such ICs is that of the therapeutic mAb,infliximab. FIG. 35 shows that infliximab+goat anti-human IgG F(ab′)2ICs induced platelet degranulation (FIG. 35, first column). The activityof this IC was blocked by mouse IV.3 mIgG2b mAb (FIG. 35, secondcolumn), by effector-deficient human MDE-8 IgG1 E269R (SEQ ID NO: 69together with SEQ ID NO: 65, FIG. 35, third column), byeffector-deficient chimeric AT-10 hIgG1 E269R (SEQ ID NO: 22 togetherwith SEQ ID NO: 16, FIG. 35, fourth column), and by effector-deficienthumanized IV.3.1 hIgG1 E269R (SEQ ID NO: 53 together with SEQ ID NO: 47,FIG. 35, fifth column). These results demonstrate that CD32a blockadecan prevent activation of platelets by ICs formed from antibodies thatcluster the therapeutic mAb, infliximab, and that the effector-deficientCD32a antibodies of the present invention are useful in methods toprevent activation of platelets by ICs, and in particular, by ICs formedfrom clusters of therapeutic non-CD32a monoclonal antibodies.

Furthermore, patients who are immunologically reactive to suchtherapeutic non-CD32a mAbs are typically transitioned, or “switched” toalternative therapeutic mAbs having the same antigen target, as is thecase in anti-TNF alpha therapy, where a reactive patient might beswitched, for example, from infliximab to adalimumab. This concern mayrequire lengthy treatment gaps to ensure that residual previoustherapeutic antibody is no longer present. However, our data suggeststhat an immune reactive recipient (i.e., a patient having an immunereaction to administered therapeutic antibodies) could safely beswitched to one of the effector-deficient CD32a antibodies describedherein with no treatment gap, and also that a patient could be treatedwith multiple effector-deficient CD32a antibodies as described hereinwithout concern for synergistic platelet activation.

Following this rationale, we injected the combination of threeanti-CD32a mAbs into CD32A transgenic mice and examined core bodytemperature and platelet counts as measures of possible synergisticinfusion reactions. Effector-deficient chimeric AT-10 hIgG1 E269R (SEQID NO: 22 together with SEQ ID NO: 16), effector-deficient chimeric IV.3hIgG2 N297A (SEQ ID NO: 51 together with SEQ ID NO: 45), andeffector-deficient human MDE-8 IgG1 E269R (SEQ ID NO: 69 together withSEQ ID NO: 65) were premixed and injected intravenously as a singlebolus into two CD32A transgenic mice. The first animal received 50micro-grams of each mAb (total of 150 micro-grams of anti-CD32a IgGinjected). The second animal received 100 micro-grams of each mAb (300micro-grams total IgG injected). Platelet counts (in whole blood) ofeach animal were measured before (FIGS. 36A and 36C) and 30 minutesafter mAb injection (FIG. 36B=50 micro-grams×3, and FIG. 36D=100micro-grams×3). Gate P1 identifies platelets (gate P4 is other bloodcells). FIG. 36E shows core body temperature in CD32A transgenic mice asmonitored for 30 minutes following injection of effector-deficient mAbs.These results show that these effector-deficient anti-CD32a clones(i.e., IV.3, AT-10, MDE-8), when combined, do not confer the capacityeither to cluster CD32a or to induce thrombocytopenia in CD32A mice.

Taken together, the results depicted by FIGS. 32-36 demonstrate asurprising functional compatability of IV.3, AT-10, and MDE-8, suchthat, when combined, these antibodies retain their non-activatingprofile, both in vitro and in vivo, thus providing a therapeuticstrategy for continued anti-CD32a immunotherapy, for example, in thepresence of anti-drug antibodies (ATA).

EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the embodiments. The foregoingdescription and Examples detail certain embodiments and describes thebest mode contemplated by the inventors. It will be appreciated,however, that no matter how detailed the foregoing may appear in text,the embodiment may be practiced in many ways and should be construed inaccordance with the appended claims and any equivalents thereof.

As used herein, the term “about” refers to a numeric value, including,for example, whole numbers, fractions, and percentages, whether or notexplicitly indicated. The term “about” generally refers to a range ofnumerical values (e.g., +/−5-10% of the recited range) that one ofordinary skill in the art would consider equivalent to the recited value(e.g., having the same function or result). In some instances, the term“about” may include numerical values that are rounded to the nearestsignificant figure.

What is claimed is:
 1. A method for inhibiting IgG-Fc ligand binding to CD32a in a human subject comprising: administering a therapeutically effective amount of an effector-deficient anti-CD32a monoclonal antibody to a human subject, wherein the antibody comprises two CD32a binding domains and at least a portion of a C_(H)2 domain, and is effector-deficient, thereby inhibiting IgG-Fc ligand binding to CD32a.
 2. The method of claim 1, wherein the effector-deficient antibody satisfies both the IgG Immune Complex Test and the Immobilized IgG Test, and wherein the FC region of the effector-deficient antibody has been altered so as to reduce or eliminate Fc-binding to CD16, CD32, and/or CD64 type IgG receptors.
 3. The method of claim 1, wherein the subject has an IgG-mediated hemostatic disorder.
 4. The method of claim 3, wherein the hemostatic disorder is thrombosis with or without thrombocytopenia.
 5. The method of claim 3, wherein the hemostatic disorder is selected from the group consisting of IgG-mediated-thrombocytopenia, immune-mediated-thrombocytopenia (ITP), antiphospholipid syndrome (APS), anti-platelet-antibody disorders, heparin-induced thrombocytopenia (HIT), and heparin-induced thrombocytopenia with thrombosis (HITT).
 6. The method of claim 1, wherein the subject has an IgG-mediated immune, autoimmune, or inflammatory disease or disorder.
 7. The method of claim 6, wherein the IgG-mediated immune, autoimmune or inflammatory disorder is selected from the group consisting of rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, ankylosing spondylitis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, antiphospholipid syndrome (APS), osteoarthritis, systemic lupus erythematous (SLE), lupus nephritis, IgG antibody-induced anemia, and IgG-mediated cytopenia.
 8. The method of claim 1, wherein the subject has an IgG immune complex-mediated disease or disorder.
 9. The method of claim 8, wherein the IgG immune complex-mediated disease or disorder is an anti-therapeutic-antibody (ATA) response caused by administration of a non-anti-CD32a monoclonal antibody or fragment thereof.
 10. The method of claim 9, wherein the non-anti-CD32a antibody is infliximab, adalimumab, certolizumab pegol (antibody-like), golimumab, etanercept (antibody-like), ustekinumab, omalizumab, or bevacizumab.
 11. The method of claim 9, wherein the effector deficient anti-CD32a antibody is administered prior to, concurrently with, or following the non-anti-CD32a monoclonal antibody.
 12. The method of claim 9, wherein the IgG immune complex-mediated disease or disorder occurs in a patient being treated with a non-anti-CD32a monoclonal antibody for the treatment of rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis, or inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease.
 13. The method of claim 1, wherein the subject has a disease or disorder characterized by IgG localized on the surface of cells circulating in the blood of the human subject.
 14. The method of claim 13, wherein the circulating cell type is comprised of one or more of the following: platelets, erythrocytes, monocytes, neutrophils, basophils, eosinophils, B-lymphocytes, macrophages, mast cells, leukemia cells, or microbes.
 15. The method of claim 13, wherein the disease or disorder is selected from one or more of the following: thrombocytopenia, leukopenia, neutropenia, lymphopenia, monocytopenia, anemia, hemolytic anemia, or sepsis.
 16. A method for treating antibody-mediated allergic or hypersensitivity reactions of type I, type II, or type III in a human subject comprising: administering a therapeutically effective amount of an effector-deficient anti-CD32a monoclonal antibody to a human subject, wherein the antibody comprises two CD32 binding domains and at least a portion of a C_(H)2 domain, and is effector-deficient, thereby treating the antibody-mediated allergic or hypersensitivity reactions of type I, type II, or type III.
 17. The method of claim 16, wherein the allergic disorder is selected from the group consisting of atopy, contact dermatitis, allergic rhinitis, systemic anaphylaxis, localized anaphylaxis as exhibited in hay fever, asthma, hives, food allergies, and eczema, allergic reactions to vaccines, allergic reactions to foods, allergic reactions to, allergic reactions to insect products, allergic reactions to drugs, allergic reactions to mold spores, allergic reactions to animal hair and dander, allergic reactions to latex, blood transfusion reactions, platelet transfusion reactions, erythrocyte transfusion reactions, erythroblastosis fetalis, hemolytic anemia, serum sickness, infusion reactions, necrotizing vasculitis, glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus, and allergic reactions to microorganisms.
 18. The method of claim 1, wherein the monoclonal antibody is humanized.
 19. The method of claim 1, wherein the antibody comprises: a. a heavy chain variable region CDR1 sequence comprising a sequence that is identical to the sequence YYWMN (SEQ ID NO: 1) or GFTFSYYW (SEQ ID NO: 73 and SEQ ID NO: 88); b. a heavy chain variable region CDR2 sequence comprising a sequence that is identical to the sequence EIRLKSNNYATHYAESVKG (SEQ ID NO: 2) or IRLKSNNYAT (SEQ ID NO: 74 and SEQ ID NO: 89); c. a heavy chain variable region CDR3 sequence comprising a sequence that is identical to the sequence RDEYYAMDY (SEQ ID NO: 3) or NRRDEYYAMDY (SEQ ID NO: 75 and SEQ ID NO: 90); d. a light chain variable region CDR1 sequence comprising a sequence that is identical to the sequence RASESVDNFGISFMN (SEQ ID NO: 4) or ESVDNFGISF (SEQ ID NO: 76 and SEQ ID NO: 91); e. a light chain variable region CDR2 sequence comprising a sequence that is identical to the sequence GASNQGS (SEQ ID NO: 5) or GAS (SEQ ID NO: 77 and SEQ ID NO: 92); and f. a light chain variable region CDR3 sequence comprising a sequence that is identical to the sequence QQSKEVPWT (SEQ ID NO: 6) or QQSKEVPWT (SEQ ID NO:78 and SEQ ID NO: 93).
 20. The method of claim 19, wherein the antibody comprises a variable heavy chain sequence comprising a sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 8 or SEQ ID NO: 12, and a variable light chain sequence comprising a sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 10 or SEQ ID NO:
 14. 21. The method of claim 19, wherein the antibody comprises: a. a heavy chain sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20; and b. a light chain sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 22, or SEQ ID NO:
 24. 22. The method of claim 1, wherein the antibody comprises: a. a heavy chain variable region CDR1 sequence comprising a sequence that is identical to the sequence NYGMN (SEQ ID NO: 25) or GYTFTNYG (SEQ ID NO: 79); b. a heavy chain variable region CDR2 sequence comprising a sequence that identical to the sequence WLNTYTGESIYPDDFKG (SEQ ID NO: 26) or LNTYTGES (SEQ ID NO: 80); c. a heavy chain variable region CDR3 sequence comprising a sequence that is identical to the sequence GDYGYDDPLDY (SEQ ID NO: 27) or ARGDYGYDDPLDY (SEQ ID NO: 81); d. a light chain variable region CDR1 sequence comprising a sequence that is identical to the sequence RSSKSLLHTNGNTYLH (SEQ ID NO: 28) or KSLLHTNGNTY (SEQ ID NO: 82 and SEQ ID NO: 100); e. a light chain variable region CDR2 sequence comprising a sequence identical to the sequence RMSVLAS (SEQ ID NO: 29) or RMS (SEQ ID NO: 83 SEQ ID NO: 101); and a light chain variable region CDR3 sequence comprising a sequence that is identical to the sequence MQHLEYPLT (SEQ ID NO: 30 and SEQ ID NO: 84 and SEQ ID NO: 102).
 23. The method of claim 22, wherein the antibody comprises: a. a variable heavy chain sequence comprising a sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 32, SEQ ID NO: 36, or SEQ ID NO: 38; and b. a variable light chain sequence comprising a sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 34, SEQ ID NO: 41 or SEQ ID
 85. 24. The method of claim 22, wherein the antibody comprises: a. a heavy chain sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 49; and b. a light chain sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 51, SEQ ID NO: 53 or SEQ ID NO:
 87. 25. The method of claim 1, wherein the antibody comprises: a. a heavy chain variable region CDR1 sequence comprising a sequence that is identical to the sequence SYGMH (SEQ ID NO: 54) or GFTFSSYG (SEQ ID NO: 94); b. a heavy chain variable region CDR2 sequence comprising a sequence that is identical to the sequence VIWYDGSNYYYTDSVKG (SEQ ID NO: 55) or IWYDGSNY (SEQ ID NO: 95); c. a heavy chain variable region CDR3 sequence comprising a sequence that is identical to the sequence DLGAAASDY (SEQ ID NO: 56) or ARDLGAAASDY (SEQ ID NO: 96); d. a light chain variable region CDR1 sequence comprising a sequence that is identical to the sequence RASQGINSALA (SEQ ID NO: 57) or QGINSA (SEQ ID NO: 97); e. a light chain variable region CDR2 sequence comprising a sequence that is identical to the sequence DASSLES (SEQ ID NO: 58) or DAS (SEQ ID NO: 98); and f. a light chain variable region CDR3 sequence comprising a sequence that is identical to the sequence QQFNSYPHT (SEQ ID NO: 59) or QQFNSYPHT (SEQ ID NO: 99).
 26. The method of claim 25, wherein the antibody comprises: a. a variable heavy chain sequence comprising a sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 61; and b. a variable light chain sequence comprising a sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO:
 63. 27. The method of claim 25, wherein the antibody comprises: a. a heavy chain sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 65 or SEQ ID NO: 67; and b. a light chain sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO:
 69. 